Methods and compositions for expressing functional class xiv myosin

ABSTRACT

The invention, in part, includes methods and compounds useful to prepare and functional class XIV myosin. Functional class XIV myosin prepared using methods of the invention may be useful to screen for and identify compounds that inhibit and treat parasitic infections and contamination.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/993,752, filed May 15, 2014 the contentof which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under USPHS grantAI054961 awarded by the National Institute of Allergy and InfectiousDisease. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention, in part, relates to methods to prepare, assess, andutilize functional class XIV myosin.

BACKGROUND OF THE INVENTION

Class XIV myosins play an important role in the life cycle ofapicomplexan parasites of medical and/or veterinary importance, such asPlasmodium spp, Toxoplasma gondii, Sarcocystis neurona andCryptosporidium parvum. One example of an apicomplexan parasite isToxoplasma gondii, which is a member of the phylum Apicomplexa and is acommon infectious agent of humans that can result in health risks toimmune-compromised individuals and the developing fetus. This obligateintracellular parasite must penetrate a host cell and replicate tosurvive. The invasive stage of the parasite relies on a unique form ofsubstrate-based motility called gliding motility, which is driven by aclass XIVa myosin motor, TgMyoA that is powered by an actomyosin-basedcomplex, which is called the “glideosome”. The class XIV myosin TgMyoAheavy chain is one of eleven myosin heavy chains found in T. gondii (1)and is an essential component of the glideosome, which is necessary forefficient parasite motility, invasion, and egress from the host.Parasites lacking TgMyoA are avirulent in a mouse model of parasiteinfection (2).

The TgMyoA motor is located between the plasma membrane and the innermembrane complex (IMC), a double membrane that is continuous around mostof the cell (3). TgGAP50 (a 50 kDa gliding associated protein), anintegral membrane glycoprotein of the IMC, acts as a membrane receptorfor the motor (4); TgMyoA is linked indirectly to TgGAP50 through anapicomplexan-specific N-terminal extension of its regulatory lightchain, TgMLC1, and TgGAP45 (a 45 kDa gliding associated protein).Several other proteins, including TgGAP40 (a 40 kDa gliding associatedprotein), TgGAP70 (a 70 kDa gliding associated protein), and TgELC1 (aputative essential light chain) have recently been identified asadditional components of this myosin motor complex (5, 6). The mechanismby which the motor complex generates motility has remained unclear.

SUMMARY OF THE INVENTION

In one aspect of the invention, methods for preparing a functional classXIV myosin are provided. Compositions that include functional class XIVmyosin polypeptides and their encoding polynucleotides are also providedin some aspects of the invention. In some aspects of the invention,methods of using functional class XIV myosin are provided and mayinclude, in some aspects, use in screening assays to identify candidatecompounds that reduce the level of activity of a class XIV myosinpolypeptide.

According to one aspect of the invention, methods of producing afunctional class XIV myosin polypeptide are provided. The methodsinclude co-expressing three or more polynucleotides in anexpression-system cell, wherein the three or more polynucleotidescomprise a class XIV heavy chain polypeptide-encoding polynucleotide, afirst myosin light chain polypeptide-encoding polynucleotide, and aparasite co-chaperone polypeptide-encoding polynucleotide, wherein thethree or more polynucleotides are co-expressed in the cell underconditions suitable to produce a functional class XIV myosin polypeptidecomprising the class XIV heavy chain polypeptide and the first myosinlight chain polypeptide. In some embodiments, the class XIV heavy chainpolypeptide-encoding polynucleotide, the parasite co-chaperonepolypeptide-encoding polynucleotide, and the first myosin light chainpolypeptide-encoding polynucleotide are each independently selected froma Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria, orCryptosporidium class XIV heavy chain polypeptide-encodingpolynucleotide or a functional variant thereof; a parasite co-chaperonepolypeptide-encoding polynucleotide or a functional variant thereof; anda myosin light chain polypeptide-encoding polynucleotide or a functionalvariant thereof, respectively. In certain embodiments, the Toxoplasma isToxoplasma gondii; Plasmodium is Plasmodium falciparum, P. vivax, P.knowlesi. P. ovale, or P. malariae; Neospora is Neospora caninu orNeospora hughesi; Sarcocystis is Sarcocystis neurona, bovihominis (S.hominis,), or S. suihominis; Eimeria is Eimeria tenella, E. bovis, E.necatrix, E. ellipsoidalis, or E. zuernii; and Cryptosporidium isCryptosporidium parvum, C. hominis, C. canis, C. felis, C. meleagridis,or C. muris. In some embodiments, co-expressing includes co-infectingthe expression-system cell with one or more expression vectors eachincluding one or more of the three or more polynucleotides. In certainembodiments, the one or more expression vectors is a viral expressionvector. In some embodiments, co-infecting the expression-system cellincludes co-infecting with one or more expression vectors comprising oneor more of the class XIV heavy chain polypeptide-encoding polynucleotideoperably linked to an independently selected promoter, the first myosinlight chain polypeptide-encoding polynucleotide operably linked to anindependently selected promoter, and the parasite co-chaperonepolypeptide-encoding polynucleotide operably linked to an independentlyselected promoter. In some embodiments, co-infecting theexpression-system cell includes co-infecting with a first expressionvector comprising the class XIV heavy chain polypeptide-encodingpolynucleotide operably linked to an independently selected promoter, asecond expression vector comprising the first myosin light chainpolypeptide-encoding polynucleotide operably linked to an independentlyselected promoter, and a third expression vector comprising the parasiteco-chaperone polypeptide-encoding polynucleotide operably linked to anindependently selected promoter. In some embodiments, the one or more ofthe expression vectors additionally comprises one or morepolynucleotides that encode: a myosin light chain-1 (MLC1) polypeptideor functional variant thereof, a tail domain interacting protein (MTIP)or functional variant thereof, an essential light chain-1, (ELC1)polypeptide or functional variant thereof, an essential like lightchain, calmodulin or a functional variant thereof, or a glideosomeassociated protein-45 (GAP45) or a functional variant thereof. In someembodiments, one or more of the expression vectors additionally compriseat least one polynucleotide sequence that encodes a detectable label. Insome embodiments, the detectable label comprises one or more FLAG tags,biotin acceptor sites, Myc tags, His tags, or Ty1 tags. In certainembodiments, the amino acid sequence of the biotin acceptor sitecomprises the sequence set forth as SEQ ID NO:21 or a functional variantthereof; the amino acid sequence of one or more of the Myc tagscomprises the sequence set forth as SEQ ID NO:22 or a functional variantthereof; and the amino acid sequence of the one or more of the Ty1 tagscomprises the sequence set forth as SEQ ID NO:23 or a functional variantthereof. In some embodiments, the parasite co-chaperonepolypeptide-encoding polynucleotide sequence comprises aUNC-45/Cro1/She4p (UCS) chaperone polynucleotide sequence, or functionalvariant thereof. In some embodiments, the parasite co-chaperonepolypeptide is derived from the sequence of a toxoplasma gondii UCS-45homolog, a Toxoplasma UNC (TgUNC) polypeptide, or a functional variantthereof. In some embodiments, the TgUNC polypeptide comprises the aminoacid sequence set forth as SEQ ID NO:12, or a functional variantthereof. In certain embodiments, the TgUNC polypeptide encoded by thepolynucleotide sequence set forth as SEQ ID NO:11, or a functionalvariant thereof. In some embodiments, the TgUNC polypeptide functionalvariant is a truncated TgUNC polypeptide. In certain embodiments, thetruncated TgUNC polypeptide comprises the amino acid sequence set forthas SEQ ID NO:14. In some embodiments, the truncated TgUNC polypeptide isencoded by the polynucleotide sequence set forth as SEQ ID NO:13. Insome embodiments, the parasite co-chaperone is derived from the sequenceof a Plasmodium falciparum UCS-45 homolog, a Plasmodium falciparum UNC(PfUNC) polypeptide, or a functional variant thereof. In certainembodiments, the PfUNC parasite co-chaperone polypeptide comprises theamino acid sequence set forth as SEQ ID NO:4, or a functional variantthereof. In some embodiments, the polynucleotide sequence of the PfUNCparasite co-chaperone comprises the sequence set forth as SEQ ID NO:3,or a functional variant thereof. In some embodiments, the PfUNCfunctional variant is a truncated PfUNC polypeptide. In certainembodiments, the truncated PfUNC polypeptide comprises the amino acidsequence set forth as SEQ ID NO:42, or a functional variant thereof. Insome embodiments, the expression system is a baculovirus/insect cellexpression system. In some embodiments, the expression-system cell is aSf9 cell. In certain embodiments, the class XIV myosin heavy chainpolypeptide comprises a TgMyoA amino acid sequence set forth as SEQ IDNO:16 or a functional variant thereof. In some embodiments, the classXIV myosin heavy chain TgMyoA amino acid sequence is encoded by thepolynucleotide sequence set forth as SEQ ID NO:15, or a functionalvariant thereof. In certain embodiments, the class XIV myosin heavychain polypeptide comprises a PfMyoA amino acid sequence set forth asSEQ ID NO:2 or a functional variant thereof. In some embodiments, theclass XIV myosin heavy chain PfMyoA amino acid sequence is encoded bythe polynucleotide sequence set forth as SEQ ID NO:1, or a functionalvariant thereof. In some embodiments, the class XIV myosin heavy chainpolypeptide is a truncated class XIV myosin heavy chain polypeptidederived from Plasmodium falciparum and comprises the amino acid sequenceset forth as SEQ ID NO:24 or a functional variant thereof, or comprisesthe amino acid sequence set forth as SEQ ID NO:26 or a functionalvariant thereof. In certain embodiments, the class XIV myosin heavychain polypeptide is a truncated class XIV myosin heavy chainpolypeptide derived from Toxoplasma gondii and comprises the amino acidsequence set forth herein as SEQ ID NO:25 or a functional variantthereof, or comprises the amino acid sequence set forth as SEQ ID NO:27or a functional variant thereof. In some embodiments, the first myosinlight chain polypeptide is a regulatory light chain (MLC1) polypeptidesequence derived from a Toxoplasma gondii regulatory light chainpolypeptide sequence, or derived from a Plasmodium falciparum regulatorylight chain polypeptide sequence. In some embodiments, the first myosinlight chain polypeptide comprises the amino acid sequence forth as SEQID NO:18 or a functional variant thereof. In some embodiments, the firstmyosin light chain polypeptide is encoded by the polynucleotide sequenceset forth as SEQ ID NO:17 or a functional variant thereof. In certainembodiments, the first myosin light chain polypeptide comprises theamino acid sequence set forth as SEQ ID NO:6 or a functional variantthereof. In some embodiments, the first myosin light chain polypeptideis encoded by the polynucleotide sequence set forth as SEQ ID NO:5 or afunctional variant thereof. In some embodiments, the method alsoincludes isolating the expressed functional class XIV myosinpolypeptide. In certain embodiments, the method also includes assayingthe function of the expressed functional class XIV myosin polypeptide.In some embodiments, the assay comprises an in vitro motility assay, abinding assay, an ATP co-sedimentation assay, or an ATPase activityassay. In some embodiments, the method also includes additionallyco-infecting the expression-system cell with an expression vectorcomprising a second myosin light chain encoding polynucleotide; andco-expressing the class XIV heavy chain polynucleotide, the first andsecond myosin light chain polynucleotides, and the parasite co-chaperonepolynucleotide under conditions suitable to produce a functional classXIV myosin polypeptide comprising the class XIV heavy chain polypeptideand the first and second myosin light chain polypeptides. In certainembodiments, the second myosin light chain polypeptide-encodingpolynucleotide encodes a Toxoplasma, Plasmodium, Neospora, Sarcocystis,Eimeria, or Cryptosporidium myosin light chain polypeptide or afunctional variant thereof. In some embodiments, the Toxoplasma isToxoplasma gondii; Plasmodium is Plasmodium falciparum, P. vivax, P.knowlesi. P. ovale, or P. malariae; Neospora is Neospora caninu orNeospora hughesi; Sarcocystis is Sarcocystisl neurona, bovihominis (S.hominis,), or S. suihominis; Eimeria is Eimeria tenella, E. bovis, E.necatrix, E. ellipsoidalis, or E. zuernii; and Cryptosporidium isCryptosporidium parvum, C. hominis, C. canis, C. felis, C. meleagridis,or C. muris. In some embodiments, the first myosin light chainpolypeptide is a regulatory light chain polypeptide and the secondmyosin light chain polypeptide is an essential light chain polypeptide.In some embodiments, the second myosin light chain is derived from aToxoplasma gondii myosin light chain sequence or a Plasmodium falciparummyosin light chain sequence. In certain embodiments, the second myosinlight chain polypeptide comprises the amino acid sequence set forth asSEQ ID NO:6, 8, 18, or 48, or a functional variant thereof. In someembodiments, the second myosin light chain polypeptide is encoded by thepolynucleotide sequence set forth as SEQ ID NO:5, 7, 17, or 47.

According to another aspect of the invention, a functional class XIVmyosin polypeptide prepared by the method of any one of the embodimentsof the aforementioned claims is provided. In some embodiments, the classXIV myosin polypeptide is an isolated functional class XIV myosinpolypeptide.

According to yet another aspect of the invention, methods of determiningan activity of a class XIV myosin polypeptide are provided. The methodsinclude (a) preparing a functional class XIV myosin polypeptide as setforth in an embodiment of any of the aforementioned methods; (b)assaying an activity of the prepared functional class XIV myosinpolypeptide; and (c) assessing the results of the assay as adetermination of activity in the functional class XIV myosinpolypeptide. In certain embodiments, the assay comprises an in vitromotility assay, a binding assay, or an ATPase activity assay. In someembodiments, the activity comprises actin movement.

According to yet another aspect of the invention, methods of identifyinga candidate compound to inhibit a parasite that expresses a class XIVmyosin polypeptide are provided. The methods including (a) preparing afunctional class XIV myosin polypeptide as set forth in an embodiment ofany of the aforementioned methods; (b) contacting the prepared class XIVmyosin polypeptide with a candidate compound under conditions suitableto determine an activity of the class XIV myosin polypeptide; (c)determining the activity of the class XIV myosin polypeptide; and (d)comparing the determined activity with a control activity determination,wherein a decrease in the determined activity in the contacted class XIVmyosin polypeptide compared to the control activity determinationidentifies the compound as a candidate compound to inhibit a parasitethat expresses the class XIV myosin polypeptide. In some embodiments,the control activity is activity determined in a functional class XIVmyosin polypeptide not contacted with the candidate compound. In certainembodiments, the determining the activity comprises determining at leastone of a level of the activity or the presence or absence of theactivity. In some embodiments, the candidate compound alters the foldingof the class XIV heavy chain polypeptide by the parasite co-chaperonepolypeptide. In certain embodiments, the candidate compound alters theinteraction between the class XIV heavy chain polypeptide and the firstlight chain polypeptide. In some embodiments, a means of determining thelevel of function of the class XIV myosin polypeptide comprises an invitro motility assay, a binding assay, or an ATPase activity assay.

According to yet another aspect of the invention, methods of producing aclass XIV myosin polypeptide are provided. The methods includingco-expressing two or more polynucleotides in an expression-system cell,wherein the two polynucleotides comprise a class XIV heavy chainpolypeptide-encoding polynucleotide and a parasite co-chaperonepolypeptide-encoding polynucleotide, wherein the two or morepolynucleotides are co-expressed in the cell under conditions suitableto produce a class XIV myosin polypeptide comprising the class XIV heavychain polypeptide. In some embodiments, the class XIV heavy chainpolypeptide-encoding polynucleotide and the a parasite co-chaperonepolypeptide-encoding polynucleotide are each independently selected froma Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria, orCryptosporidium class XIV heavy chain polypeptide or a functionalvariant thereof; and a from a Toxoplasma, Plasmodium, Neospora,Sarcocystis, Eimeria, or Cryptosporidium parasite co-chaperonepolypeptide-encoding polynucleotide or a functional variant thereof. Incertain embodiments, the method also includes co-expressing an essentialmyosin light chain polypeptide-encoding polynucleotide independentlyselected from a Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria,or Cryptosporidium essential myosin light chain-encoding polynucleotideor a functional variant thereof. In some embodiments, the Toxoplasma isToxoplasma gondii; Plasmodium is Plasmodium falciparum, P. vivax, P.knowlesi. P. ovale, or P. malariae; Neospora is Neospora caninu orNeospora hughesi; Sarcocystis is Sarcocystisl neurona, bovihominis (S.hominis,), or S. suihominis; Eimeria is Eimeria tenella, E. bovis, E.necatrix, E. ellipsoidalis, or E. zuernii; and Cryptosporidium isCryptosporidium parvum, C. hominis, C. canis, C. felis, C. meleagridis,or C. muris.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. The present invention is not intended to be limitedto a system or method that must satisfy one or more of any statedobjects or features of the invention. The present invention is notlimited to the exemplary or primary embodiments described herein.Modifications and substitutions by one of ordinary skill in the art areconsidered to be within the scope of the present invention. Also, thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of‘including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

Partial Listing of SequencesSEQ ID NO: 1 Plasmodium falciparum 3D7 myosin A (MyoA) mRNA, complete cds,nucleotide sequence: XM_001350111, (also referred to hereinas PfMyoA heavy chain)atggctgttacaaatgaagaaataaaaacggcaagtaagattgttagaagagtttcaaatgtagaagcatttgacaaaagtggttcagtttttaagggttatcaaatatggactgatatatctccgacaatagaaaatgatccaaatattatgtttgtaaaatgtgttgtacaacaaggatcaaaaaaagaaaaattaaccgttgtacaaattgatccacccggaacaggaactccatacgatattgatccaactcacgcatggaactgcaactctcaagtagaccccatgtcttttggtgatattggtcttttaaatcacaccaacatcccatgtgttcttgactttttaaagcacagatatttaaaaaatcaaatatacaccactgctgttccccttattgttgcaataaacccatacaaggatttaggaaacacaactaatgaatggattcgtagatatcgtgatacagctgatcatactaagttgccaccacacgtgttcacatgtgctagggaagctttgtctaatctccatggtgtaaacaagagccaaactattattgtatctggtgaatctggtgcaggaaaaaccgaagcaacaaaacaaatcatgagatattttgcttcttctaagagtggaaatatggatttacgtattcagacagcaataatggctgcaaatccagttcttgaagcttttggtaatgcgaaaactataagaaataacaattcatctcgttttggtcgtttcatgcagttggttatatcccatgaaggaggtataagatacggttccgttgttgcttttctgttggaaaaatctagaattattacacaagatgataatgaaaggtcatatcatatattttatcaatttcttaagggtgcaaatagtacgatgaaatctaaatttggtttaaaaggagttactgaatacaaattattgaacccaaattcaacagaggtaagtggagtagatgatgtaaaagattttgaagaggtaattgaatcgttgaaaaatatggaattaagtgaatcagatattgaagtaatattttcaatagtagctggtatattaacattaggaaatgtaagattaattgagaagcaagaagctggattaagtgatgctgctgctattatggatgaggatatgggtgtgtttaataaagcttgtgaattgatgtatttagaccctgaattaataaaaagggaaatattaattaaggtaactgttgctggaggaacaaaaattgaaggtagatggaataaaaatgatgcagaagtgttgaaatcttccttatgtaaagctatgtatgagaaattgtttttatggataataagacatttgaattcaagaattgaaccagaaggaggatttaaaacatttatgggtatgttagatatttttggttttgaagtatttaaaaataattcattggaacaattatttattaacattactaacgaaatgcttcagaaaaattttgtagatattgtttttgaaagagaatcaaaattatataaagacgaaggaatatcaacagctgaattaaagtacaccagtaataaggaagtaataaacgtactttgtgagaagggtaaatcagtactttcatacttagaggaccaatgtttagcacctggaggaaccgatgaaaagtttgtaagttcctgtgctacaaatttaaaggaaaataataagtttaccccagcaaaagtagcatcgaataaaaattttataatacaacatactataggaccaattcaatattgtgctgaaagctttttgcttaaaaacaaggatgtcttaagaggtgatttagttgaagtaattaaggattcccccaatccaatagtacaacagttatttgaaggtcaagtaattgagaagggtaaaatagctaaaggttcattaataggttctcaatttttaaatcaattgacatctttaatgaacttgataaatagtactgaaccacatttcatacgttgtattaaaccaaatgaaaataaaaaaccattagaatggtgtgaaccaaaaatattaattcagcttcatgccttatcaattttagaagcattagtattaagacaattaggatattcttatagaagaacctttgaagaattcttatatcaatataaatttgtggacattgctgctgctgaagattcatcagttgaaaaccaaaataaatgtgttaatatattaaagttgtctggactatctgaatccatgtataagataggaaaaagcatggtctttttgaaacaagaaggtgcaaaaatattgacaaaaatacaaagagagaaacttgttgaatgggaaaattgtgtgagtgtaattgaagctgctatacttaaacacaaatacaaacaaaaggttaacaaaaatataccttctcttttgagagtacaagctcatataagaaaaaaaatggtagctcaataa.SEQ ID NO: 2 Plasmodium falciparum 3D7 myosin A (MyoA), amino acid sequence:XP_001350147.1, (also referred to herein as PfMyoA heavy chain)MAVTNEEIKTASKIVRRVSNVEAFDKSGSVFKGYQIWTDISPTIENDPNIMFVKCVVQQGSKKEKLTVVQIDPPGTGTPYDIDPTHAWNCNSQVDPMSFGDIGLLNHTNIPCVLDFLKHRYLKNQIYTTAVPLIVAINPYKDLGNTTNEWIRRYRDTADHTKLPPHVFTCAREALSNLHGVNKSQTIIVSGESGAGKTEATKQIMRYFASSKSGNMDLRIQTAIMAANPVLEAFGNAKTIRNNNSSRFGRFMQLVISHEGGIRYGSVVAFLLEKSRIITQDDNERSYHIFYQFLKGANSTMKSKFGLKGVTEYKLLNPNSTEVSGVDDVKDFEEVIESLKNMELSESDIEVIFSIVAGILTLGNVRLIEKQEAGLSDAAAIMDEDMGVFNKACELMYLDPELIKREILIKVTVAGGTKIEGRWNKNDAEVLKSSLCKAMYEKLFLWIIRHLNSRIEPEGGFKTFMGMLDIFGFEVFKNNSLEQLFINITNEMLQKNFVDIVFERESKLYKDEGISTAELKYTSNKEVINVLCEKGKSVLSYLEDQCLAPGGTDEKFVSSCATNLKENNKFTPAKVASNKNFIIQHTIGPIQYCAESFLLKNKDVLRGDLVEVIKDSPNPIVQQLFEGQVIEKGKIAKGSLIGSQFLNQLTSLMNLINSTEPHFIRCIKPNENKKPLEWCEPKILIQLHALSILEALVLRQLGYSYRRTFEEFLYQYKFVDIAAAEDSSVENQNKCVNILKLSGLSESMYKIGKSMVFLKQEGAKILTKIQREKLVEWENCVSVIEAAILKHKYKQKVNKNIPSLLRVQAHIRKKMVAQ.SEQ ID NO: 3 Plasmodium falciparum 3D7 Tetratricopeptide repeat family protein(PFUNC), putative (PF14_0196) Nucleotide sequence: XM_001348333 mRNA, CDS (alsoreferred to herein as PFUNC, PFUNC and/or PfUnc)atgcaggaatttgttatgaatattcttagtaaagaaaaaattgggaaaatagaaaaactaaaaaatgatgggaatgaattatatcgaaataaaaaatataaggaggctttatgtatatatgataatgctgtttgtgagttttgtggaggttcaggtgatagtttaaaaattaaggaaatggtgaaagaattttcaaaaaatgatgagactaataatataaataaattatcagatgataataataataataatatgattgatgaaaataataagaaatgtgatttttcttatatatgtgaaattcaaaatttattcataaaaatatgccataatatatcattatgttattatttcttagaagattttgaaaaatctattgaatattgtttatatattaatgaaatgaacaataatcattataaaagttaccacacgcttgggttgtgttatgagaaattaaaagattatcaaaagagtatacattattttgataggtgtaaaatagtacttttaaaaaatcaaaacaataaaaataaagacaataataataaaagtgaaattaatcgaattaatgaaaagttacgtgatattatgaaaattatagatcaaaataaaaatgatccatataaaaatataagcaacattaaaaaatatcttcttgatgaaaatacaaataatataaatgacattgaagaaaataataaaaaaattaaattgttacattctatttataatcaaaagttttatatactacttaaggaaaatatttttttatttctttttgattttattaaaaaaaataatgacatatcaaactatgacgattgcaataataataataataataataatttatatagtcataatattaatagtttattattagaaaaaacagccatttatgttatttataaaatattatccaaattagataatgaacatattattattgaaaattccaaagatgataactataataaaaatcgtaatatatgtattaacaaattagataattcaaagcttcaatattattatgatctagattatattaaaatgattttatcctttaatgaatattttacaaaagattggatatataattatataaaaaaaaaaatcaatatattagaaaatttaaagttttcaaaagatgaaaccttatataaagaacatgtagacattttaatctatatcataaatattatgaaatatgtctatgttataaataatgattatattttaaatattattaattcctattatttaaatagcgataattctaatattaataactctggtattaatgctcttacattcttatgtaaaaaaaaacaatttttaacacaaaatagtaaggataatagaaaaaaaaaaaatgatttattagaaatgcttaaaaaagaaaattacttaaatatacaaaataatatacaaaataataataataataataattattatttttataagaatgaatttattgaatttcacttttcggactctcataaatatccattgtgtatcaattccgaaatcaaaaaaattatacaaaatgttattggtatgtatgaacacttttctagtagtatagaatataccttaattttaatttttactttattacatgacccacaaagacctaaggaaaaagacatagaaatgaatgatgtaatttatgattgtatagataattattttcatcataatgaaaatattttgatagaatggtttgtatgtattaaatgtcttttcttagtagataaaaatattatattaaattatttaataggaaaaacagaatatattgtaaaaatcttacattttattacgaactgtataggaagaaaaactaaagaagaattatccatatatatcgatgtcttattacttttattgaatatatcagaaatacgttttatgtttactaattatattgatatgtatataaatataatgaaatctttaaattatgatcaatgctttttgaaacttttactaggtacatttaaattatacatgcacaacatagactttaaacaacaaattcaagataatgtggatttgttcttttatgcgaaagaaatattaaagcaattcttattaacatatgataatgatgcggacggaaaaaataacacgaataatgataaaagagaagaaaatgaggaacagacaagccatttgaattattctaatttaaattcgtacacatgctgtgtgaaaaaatgtgatgataatgatacaaagaaaaaagatattataaaccaaaagaaagatgaaaagaataagaaacattgtgaacttgtagataagaaaaaaaaagatcatacatatatacattcgaatatgagttgtgaaaaaacgttaaaagatttaatagaaatgttattttatttaagtttgcatattgaatttaaaaaacaattattagaggaaaaaaataattatattttatttttcttaataaaagttggacatgatataaataaaaagaaattagataacacatataaatatatatattgcaacactataaataatttaattttaacaaaaaatgatgaaaaaataaaaagaagagaaattaataaaactaatttatcaaattttgataatgaacaaatagaagctttagaacaattttatgataaattaccaaaagaagctagacctaaaacagatccattatatgattatggagatgaagaaacaagtaacaaattaattgatttattattatataatgaaaaatatcaaatgaatcatattaatgataaaaataaaaataataataataataataatattaataatggtaacgtatctcctttgtcatctaaatgttcctatacaaatggtaccattatcaatattatatataactttattaatagcaatttttttacaacaaatatagctgaatctgtatgtgaaataatttcaaagtttgttaaaaatacaaataatattggtatagttttagttaataatggattaaaaactttattattagcatctaagcatataacaaataaaaagaattgtgctttagcattaagtgaaatatttatttatacgaacccgaaacttattcatttttatgaagcatatgattctttacctttattaattgaacaactaaagagtgatgaagaattattaatctttaaaacgttaatggcaataactaatattttaactattgatgaaaatgtagcaataaaagctatgcaattaaatttatggtataaatgttttgatattctttcaacagaaaatgaatacataaaatctgctagcttagaatgtatatgcaatttatgttcccaatcacatgtacatcaatatatttatgataaatatcaaacaattatgaaatcaaaaaatgaatcagataaagatattttatttgttgatattcaaataatttattcatttaccatggaatatcaaaattataaatgtgtttttgcagcaactggagctttaggtatgttgtcatctgatttgcgtttgccatattatttagttagaactaaagggattgatcatattttctcatctttcaataataccaccgaccaaaatattttattacgtattttaacatttttcaacaacataatgacgtgtgatgatataccggatgatatattaaagaaaataaagacttatgtggagaaaaagaaggatttaaatgaagagaatactcaaatggcaaattttatactccagtag.SEQ ID NO: 4 Plasmodium falciparum 3D7 Tetratricopeptide repeat familyprotein, putative (PF14_0196) Amino acid sequence XP_001348369.1(also referred to herein as PFUnc, PfUNC, and/or PFUNC)MQEFVMNILSKEKIGKIEKLKNDGNELYRNKKYKEALCIYDNAVCEFCGGSGDSLKIKEMVKEFSKNDETNNINKLSDDNNNNNMIDENNKKCDFSYICEIQNLFIKICHNISLCYYFLEDFEKSIEYCLYINEMNNNHYKSYHTLGLCYEKLKDYQKSIHYFDRCKIVLLKNQNNKNKDNNNKSEINRINEKLRDIMKIIDQNKNDPYKNISNIKKYLLDENTNNINDIEENNKKIKLLHSIYNQKFYILLKENIFLFLFDFIKKNNDISNYDDCNNNNNNNNLYSHNINSLLLEKTAIYVIYKILSKLDNEHIIIENSKDDNYNKNRNICINKLDNSKLQYYYDLDYIKMILSFNEYFTKDWIYNYIKKKINILENLKFSKDETLYKEHVDILIYIINIMKYVYVINNDYILNIINSYYLNSDNSNINNSGINALTFLCKKKQFLTQNSKDNRKKKNDLLEMLKKENYLNIQNNIQNNNNNNNYYFYKNEFIEFHFSDSHKYPLCINSEIKKIIQNVIGMYEHFSSSIEYTLILIFTLLHDPQRPKEKDIEMNDVIYDCIDNYFHHNENILIEWFVCIKCLFLVDKNIILNYLIGKTEYIVKILHFITNCIGRKTKEELSIYIDVLLLLLNISEIRFMFTNYIDMYINIMKSLNYDQCFLKLLLGTFKLYMHNIDFKQQIQDNVDLFFYAKEILKQFLLTYDNDADGKNNTNNDKREENEEQTSHLNYSNLNSYTCCVKKCDDNDTKKKDIINQKKDEKNKKHCELVDKKKKDHTYIHSNMSCEKTLKDLIEMLFYLSLHIEFKKQLLEEKNNYILFFLIKVGHDINKKKLDNTYKYIYCNTINNLILTKNDEKIKRREINKTNLSNFDNEQIEALEQFYDKLPKEARPKTDPLYDYGDEETSNKLIDLLLYNEKYQMNHINDKNKNNNNNNNINNGNVSPLSSKCSYTNGTIINIIYNFINSNFFTTNIAESVCEIISKFVKNTNNIGIVLVNNGLKTLLLASKHITNKKNCALALSEIFIYTNPKLIHFYEAYDSLPLLIEQLKSDEELLIFKTLMAITNILTIDENVAIKAMQLNLWYKCFDILSTENEYIKSASLECICNLCSQSHVHQYIYDKYQTIMKSKNESDKDILFVDIQIIYSFTMEYQNYKCVFAATGALGMLSSDLRLPYYLVRTKGIDHIFSSFNNTTDQNILLRILTFFNNIMTCDDIPDDILKKIKTYVEKKKDLNEENTQMANFILQ.SEQ ID NO: 5 Plasmodium falciparum 3D7 myosin A tail domain interactingprotein (MTIP) mRNA, complete cds, nucleotide sequence: XM_001350813.1(referred to herein as Pf MTIP, Plasmodium falciparum MLC1,or Plasmodium falciparum regulatory light chain)atgaaacaagaatgcaatgtatgttattttaacttgcctgacccagagtccaccttaggtccatatgataatgaattaaattatttcacttggggaccaggatttgaatatgaacctgaaccacaaagaaagccattgtcaattgaagaaagttttgaaaactctgaagaatccgaagaatcagttgctgacatacaacaacttgaagaaaaagtagatgaaagtgatgtgaggatttattttaatgaaaagagtagtggtgggaaaataagtatagacaatgcatcttacaatgctcgaaagttaggtttagctccatcaagtatcgatgaaaagaaaattaaagaattatatggagataacttaacatatgaacaatatttagaatatttgtctatatgtgtccatgataaagataatgtagaagaacttattaaaatgtttgcacactttgataataattgtactggttacttaactaagagccaaatgaaaaatattcttacaacttggggtgatgcattaacggatcaagaagccatagatgctcttaatgccttttcatcagaagataacattgattacaaattattctgtgaagatatattacaataa.SEQ ID NO: 6 Plasmodium falciparum 3D7 myosin A tail domain interacting protein(MTIP) amino acid sequence: XP_001350849.1 (also referred to herein as Pf MTIP,Plasmodium falciparum MLC1, or Plasmodium falciparum regulatory light chain)MKQECNVCYFNLPDPESTLGPYDNELNYFTWGPGFEYEPEPQRKPLSIEESFENSEESEESVADIQQLEEKVDESDVRIYFNEKSSGGKISIDNASYNARKLGLAPSSIDEKKIKELYGDNLTYEQYLEYLSICVHDKDNVEELIKMFAHFDNNCTGYLTKSQMKNILTTWGDALTDQEAIDALNAFSSEDNIDYKLFCEDILQ .SEQ ID NO: 7 Plasmodium falciparum 3D7 calmodulin, putative (PF14 0181) mRNA,complete cds, nucleotide sequence: XM 001348318.1 (also referred toherein as PfELC or Plasmodium falciparum essential light chain)atgcgcatagtagataagcaaataaaagaatccttccttttagcagacagaaattttgatgggcatatttcatcgaatgaattattatacgctttaagattccttggagtggaatctgattattctctaatggaaaataaaagtggggcaacttattcaatgaatgattatgttaaaatagctaagaaacatttaggtgcacacacaccaaaagaaagaattacaaattccttaaaaaaaatggataaaaataacaatggaactatatcagttgacgcattagttcatttagttatgactatgagtgatattttaacagaaaatgattacagaaaatttaaaaaatttgttgatcctgaaagcagaaatatcataccgttacatgtatttgtagaaaaaatactttcgtaa.SEQ ID NO: 8 Plasmodium falciparum 3D7 calmodulin, putative (PF14 0181),amino acid sequence: XP_001348354.1 (also referred to herein as PfELC (alsoreferred to herein as PfELC or Plasmodium falciparum essential light chain)MRIVDKQIKESFLLADRNFDGHISSNELLYALRFLGVESDYSLMENKSGATYSMNDYVKIAKKHLGAHTPKERITNSLKKMDKNNNGTISVDALVHLVMTMSDILTENDYRKFKKFVDPESRNIIPLHVFVEKILS .SEQ ID NO: 9 Plasmodium falciparum 3D7 calmodulin, putative (PF14_0181) mRNA,complete cds, nucleotide sequence: XM_001348318.1 (also referred to herein asPfCalmodulin)atgcgcatagtagataagcaaataaaagaatccttccttttagcagacagaaattttgatgggcatatttcatcgaatgaattattatacgctttaagattccttggagtggaatctgattattctctaatggaaaataaaagtggggcaacttattcaatgaatgattatgttaaaatagctaagaaacatttaggtgcacacacaccaaaagaaagaattacaaattccttaaaaaaaatggataaaaataacaatggaactatatcagttgacgcattagttcatttagttatgactatgagtgatattttaacagaaaatgattacagaaaatttaaaaaatttgttgatcctgaaagcagaaatatcataccgttacatgtatttgtagaaaaaatactttcgtaa.SEQ ID NO: 10 Plasmodium falciparum 3D7 calmodulin, puatative (PF13_0181)amino acid sequence: XP_001348497 (also referred to herein as PfCalmodulin)MADKLTEEQISEFKEAFSLEFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEIDTDGNGTIDFPEFLTLMARKLKDTDTEEELIEAFRVFDRDGDGYISADELRHVMTNLGEKLTNEEVDEMIREADIDGDGQINYEEFVKMMIAK.SEQ ID NO: 11 Toxoplasma gondii TPR AK Seq, TPR domain-containing protein, CDS,length = 3486 nucleotide sequence (also referred to herein as TgUNC)atggaggatttgtcaaacgctacgttagctcgcctccaggcgctgaaagaggaaggaaatgcggagtttaagcggggcaagttcgagtccgcgatcgaagcgtattcgcgatgcctggctgatgcctcagacactctggataaagaaccagacgtcctgggaggcgcgtgtgcggccagcctctcttcctccgactctcaggtcgcagaacctcgcaaagaaagccctgccattctgaagcgcgtggccgaactgaaggcgcaaatcctgtgcaatcgcgccctgtgctaccagcggacgaagcagttcgcggcggccgaggcggactgtacgcgcgccatcgctctccatccggcctacgtgaagagttactaccgacgcgcggttgcgctggacgcccagggaaggcgtaaagagtgtgtggaggatctgcagacgtgtctgcgcctgcagcctggaaacaaggaggcgcaggagatgctcgcgggggttcgcgacaaggtgatgaaggaggaggagacgcgcgtcgaagagcagctgcccgagaacctgctgactgccggcctcaacgacgtgctcacggcctcgaagcgagtcgcctcccttcgccaactcggcgccttcgttcaggagcggaagctgcggcgtcagttccttcgagacggcggtctgcgacgcgttgccgttgccctgaagagccaaatggacagtgagctcgagggcaacggccagcctcaccggtcgctcgagtctcttccggagaccacggcctcggaccccgtggcttctgaagagagcaaaagcagcgcagacgccgttctccccgtcagcgtcgaagcagcgtgctgggagttgctcttgtccgtcgtccagcagcaccaggcggacgacgaagacgacgcaaacaaggctctgcagacctctgtggaggccgtgaacgcgccgcttcaggtcgacgcgccggtcctcgagtgccgccaagctctctccggcctctggactcccaacgacttcctcgtgcgtctgcggcagctcctgcgtgcaggcgtcgcgcgctccgacggcctcgctgcgaccgccgcgaacgaagaagctgggaaactgcggacctgcgtgcaggcgtcgcgcgctccgacggcctcgctgcgaccgccgcgaacgaagaagctgggaaactgcggaccgtctggcgcggcgaagcctgtgacaggctcctgcgcacgatgggctgcgtggtgcagctgcaggcggcgcagttcgacgaggacgcctccttcctcgaggcctgcgccgccggcctggagtgtatagactcccgagaggtccagcgcgcggctgtcgcggcgcttgtgggcgtcgcagacgcgcggcgccgcctgggcggcagagttgcggccgtgcggctgcggcatggactcgaaaagtgtctcgaggacgccttgcaggtcgtctcggacgcagaacacgaactcgcgggcgaagacgctcgctctcagctggccaccgcgtccaagaaaagcgacagacttgaagcggtgagtcgtctacagggccagaccgagtacttgatcatcacgctaatcgctcttctcgcagacaaagaccgcggcaaggaggagcctcccgacatgagtcgcctggtcgaccagctgctgtcgccgtacttccggccgtgcgcagaccccgaggagagcgtcgtcaccctgacagttgggttgaaggcgctgcgtttgattctgactgcggcgcgcgaagttgcgcgggcgtacctgatttccgcgtcttcgattctcccctacctcctggctgccgccgcgggcggtgtaggtacagcgcagagcggggccgcggggactgcagcgcatcggcgccagcaggaagctgcattggaggtcttgctggcctgcatggatttcccagaactgcgtgcgacgttgctcgaggcgaacgcggtgccggtcttcgcgaaagtgtgcagcgagagcgcgaatgtcgggtgttggatgagggcgcgtctggcggctgcactcgctcggctgtctgttcacgacgaggacgtccggatccaggtatttgactcgatcgacttctacgacgtcctcgatgtgctcgtgtcggaaattcgcgcggcaggtggcgacggccgccaggttgcggagccgagcaccgaagccaagaacgcgcagaaaggccagccgatggctgttggagaggagacgtttcggtctctgcttgagatcttcttcttcctcagtctccacggcgacttcaaagcgcggctcgtgaccgggaagaagggcgcgagggtgctgcggacgcttctgcaagttgcgaacggggctgggaaaaaaggcgcttcttcgagtctgacgcggtatctccttctccagagtctgtgcaacatcatgcggtcgcgagaagaccgacagagacagcggagaaggaaaggcgaggtcggaagtccactcgcagacgttgacgacgagcagctgcagcagctcgaggagttgttcaagaagctgccggaaggcgcgaagccggctgcgaacggcgaggtcgatctcggagacaaggccctcgcaacgcagctccgcgacatgctcctagacctgaacgtggttcatgcaatcgccgtgaacgtctgcgcgaccccgccgccgtcttccaacgtcctgtgtgccgcggcgcaggcgctgaagttcctttgcgaggactcgcggcaccgaggcagagctgttcaggaggggggcattcggacgctcttggtggccgcgagtgggctcgaggaattcccagacgaccagaggaacgcgcgacaagctgcggcgcagctctgcatcaccaccaacccggccctgttctcttaccgcgagagcttagacctcgtcccctgcctcgcgccgcttctcaaggaccgccacgagttgctgcagtacgaaggcgcgctcgcgttgacgaatctctgtgccctcagcgaggaggtccgcatgcgtgcgtggctcggcggcgtctgggaaggcttcgaggacctcatgttcggggagaacgagttgctgcgcgccgcggggttggagggatggtgcaacttgtcggcctcgccgacggtccaaacggagatcgggaagaagatggaacgtttcgcggcggagaaacaagaagttcaagatatgaaactcttgctcgcgttcacgcgggaaacgaacaaccctcgtgcgcagtccgccgccgtcgcggctctcgcgatgcttctcgcgaacgagaaggtcgcacgctgtcttccggcctacagcctctttggcaacctcgctttgagcctcgaggaagcgaaggccgagcaggaagctctgatcgtgaggtgcgtctccgccctctacaacgtctggatcgagttgagcagttctgaggcgggagctgagacccgcatgcagatcgtgaaaaccctgcagagaaaccaacaaaaactcactggagacgcacaacacctcgccaaggaagtcctcacggcagaactctcccaagcaaacacacacacgaaagaatccaccccagactcaagctaacggc.SEQ ID NO: 12 TGGT1_249480, Toxoplasma gondii GT1, tetratricopeptide repeat-containing protein, length =1161, amino acid sequence, (also referred to herein as TgUNC)MEDLSNATLARLQALKEEGNAEFKRGKFESAIEAYSRCLADASDTLDKEPDVLGGACAASLSSSDSQVAEPRKESPAILKRVAELKAQILCNRALCYQRTKQFAAAEADCTRAIALHPAYVKSYYRRAVALDAQGRRKECVEDLQTCLRLQPGNKEAQEMLAGVRDKVMKEEETRVEEQLPENLLTAGLNDVLTASKRVASLRQLGAFVQERKLRRQFLRDGGLRRVAVALKSQMDSELEGNGQPHRSLESLPETTASDPVASEESKSSADAVLPVSVEAACWELLLSVVQQHQADDEDDANKALQTSVEAVNAPLQVDAPVLECRQALSGLWTPNDFLVRLRQLLRAGVARSDGLAATAANEEAGKLRTVWRGEACDRLLRTMGCVVQLQAAQFDEDASFLEACAAGLECIDSREVQRAAVAALVGVADARRRLGGRVAAVRLRHGLEKCLEDALQVVSDAEHELAGEDARSQLATASKKSDRLEAVSRLOGOTEYLIITLIALLADKDRGKEEPPDMSRLVDQLLSPYFRPCADPEESVVTLTVGLKALRLILTAAREVARAYLISASSILPYLLAAAAGGVGTAQSGAAGTAAHRRQQEAALEVLIACMDFPELRATLLEANAVPVFAKVCSESANVGCWMRARLAAALARLSVHDEDVRIQVFDSIDFYDVLDVLVSEIRAAGGDGRQVAEPSTEAKNAQKGQPMAVGEETFRSLLEIFFFLSLHGDFKARLVTGKKGARVLRTLLQVANGAGKKGASSSLTRYLLLQSLCNIMRSREDRQRQRRRKGEVGSPLADVDDEQLQQLEELFKKLPEGAKPAANGEVDLGDKALATQLRDMLLDLNVVHAIAVNVCATPPPSSNVLCAAAQALKFLCEDSRHRGRAVQEGGIRTLLVAASGLEEFPDDQRNARQAAAQLCITTNPALFSYRESLDLVPCLAPLLKDRHELLQYEGALALTNLCALSEEVRMRAWLGGVWEGFEDLMFGENELLRAAGLEGWCNLSASPTVQTEIGKKMERFAAEKQEVQDMKLLLAFTRETNNPRAQSAAVAALAMLLANEKVARCLPAYSLFGNLALSLEEAKAEQEALIVRCVSALYNVWIELSSSEAGAETRMQIVKTLQRNQQKLTGDAQHLAKEVLTAELSQANTHTKESTPDSS.SEQ ID NO: 13 Truncated Toxoplasma gondii TgUNC nucleotide sequence, CDS:atgggactcgcgggggttcgcgacaaggtgatgaaggaggaggagacgcgcgtcgaagagcagctgcccgagaacctgctgactgccggcctcaacgacgtgctcacggcctcgaagcgagtcgcctcccttcgccaactcggcgccttcgttcaggagcggaagctgcggcgtcagttccttcgagacggcggtctgcgacgcgttgccgttgccctgaagagccaaatggacagtgagctcgagggcaacggccagcctcaccggtcgctcgagtctcttccggagaccacggcctcggaccccgtggcttctgaagagagcaaaagcagcgcagacgccgttctccccgtcagcgtcgaagcagcgtgctgggagttgctcttgtccgtcgtccagcagcaccaggcggacgacgaagacgacgcaaacaaggctctgcagacctctgtggaggccgtgaacgcgccgcttcaggtcgacgcgccggtcctcgagtgccgccaagctctctccggcctctggactcccaacgacttcctcgtgcgtctgcggcagctcctgcgtgcaggcgtcgcgcgctccgacggcctcgctgcgaccgccgcgaacgaagaagctgggaaactgcggaccgtctggcgcggcgaagcctgtgacaggctcctgcgcacgatgggctgcgtggtgcagctgcaggcggcgcagttcgacgaggacgcctccttcctcgaggcctgcgccgccggcctggagtgtatagactcccgagaggtccagcgcgcggctgtcgcggcgcttgtgggcgtcgcagacgcgcggcgccgcctgggcggcagagttgcggccgtgcggctgcggcatggactcgaaaagtgtctcgaggacgccttgcaggtcgtctcggacgcagaacacgaactcgcgggcgaagacgctcgctctcagctggccaccgcgtccaagaaaagcgacagacttgaagcggtgagtcgtctacagggccagaccgagtacttgatcatcacgctaatcgctcttctcgcagacaaagaccgcggcaaggaggagcctcccgacatgagtcgcctggtcgaccagctgctgtcgccgtacttccggccgtgcgcagaccccgaggagagcgtcgtcaccctgacagttgggttgaaggcgctgcgtttgattctgactgcggcgcgcgaagttgcgcgggcgtacctgatttccgcgtcttcgattctcccctacctcctggctgccgccgcgggcggtgtaggtacagcgcagagcggggccgcggggactgcagcgcatcggcgccagcaggaagctgcattggaggtcttgctggcctgcatggatttcccagaactgcgtgcgacgttgctcgaggcgaacgcggtgccggtcttcgcgaaagtgtgcagcgagagcgcgaatgtcgggtgttggatgagggcgcgtctggcggctgcactcgctcggctgtctgttcacgacgaggacgtccggatccaggtatttgactcgatcgacttctacgacgtcctcgatgtgctcgtgtcggaaattcgcgcggcaggtggcgacggccgccaggttgcggagccgagcaccgaagccaagaacgcgcagaaaggccagccgatggctgttggagaggagacgtttcggtctctgcttgagatcttcttcttcctcagtctccacggcgacttcaaagcgcggctcgtgaccgggaagaagggcgcgagggtgctgcggacgcttctgcaagttgcgaacggggctgggaaaaaaggcgcttcttcgagtctgacgcggtatctccttctccagagtctgtgcaacatcatgcggtcgcgagaagaccgacagagacagcggagaaggaaaggcgaggtcggaagtccactcgcagacgttgacgacgagcagctgcagcagctcgaggagttgttcaagaagctgccggaaggcgcgaagccggctgcgaacggcgaggtcgatctcggagacaaggccctcgcaacgcagctccgcgacatgctcctagacctgaacgtggttcatgcaatcgccgtgaacgtctgcgcgaccccgccgccgtcttccaacgtcctgtgtgccgcggcgcaggcgctgaagttcctttgcgaggactcgcggcaccgaggcagagctgttcaggaggggggcattcggacgctcttggtggccgcgagtgggctcgaggaattcccagacgaccagaggaacgcgcgacaagctgcggcgcagctctgcatcaccaccaacccggccctgttctcttaccgcgagagcttagacctcgtcccctgcctcgcgccgcttctcaaggaccgccacgagttgctgcagtacgaaggcgcgctcgcgttgacgaatctctgtgccctcagcgaggaggtccgcatgcgtgcgtggctcggcggcgtctgggaaggcttcgaggacctcatgttcggggagaacgagttgctgcgcgccgcggggttggagggatggtgcaacttgtcggcctcgccgacggtccaaacggagatcgggaagaagatggaacgtttcgcggcggagaaacaagaagttcaagatatgaaactcttgctcgcgttcacgcgggaaacgaacaaccctcgtgcgcagtccgccgccgtcgcggctctcgcgatgcttctcgcgaacgagaaggtcgcacgctgtcttccggcctacagcctctttggcaacctcgctttgagcctcgaggaagcgaaggccgagcaggaagctctgatcgtgaggtgcgtctccgccctctacaacgtctggatcgagttgagcagttctgaggcgggagctgagacccgcatgcagatcgtgaaaaccctgcagagaaaccaacaaaaactcactggagacgcacaacacctcgccaaggaagtcctcacggcagaactctcccaagcaaacacacacacgaaagaatccaccccagactcaagctaa.SEQ ID NO: 14 Truncated Toxoplasma gondii TgUNC amino acid sequence:MGLAGVRDKVMKEEETRVEEQLPENLLTAGLNDVLTASKRVASLRQLGAFVQERKLRRQFLRDGGLRRVAVALKSQMDSELEGNGQPHRSLESLPETTASDPVASEESKSSADAVLPVSVEAACWELLLSVVQQHQADDEDDANKALQTSVEAVNAPLQVDAPVLECRQALSGLWTPNDFLVRLRQLLRAGVARSDGLAATAANEEAGKLRTVWRGEACDRLLRTMGCVVOLQAAQFDEDASFLEACAAGLECIDSREVQRAAVAALVGVADARRRLGGRVAAVRLRHGLEKCLEDALQVVSDAEHELAGEDARSQLATASKKSDRLEAVSRLQGQTEYLIITLIALLADKDRGKEEPPDMSRLVDQLLSPYFRPCADPEESVVTLTVGLKALRLILTAAREVARAYLISASSILPYLLAAAAGGVGTAQSGAAGTAAHRRQQEAALEVLLACMDFPELRATLLEANAVPVFAKVCSESANVGCWMRARLAAALARLSVHDEDVRIQVFDSIDFYDVLDVLVSEIRAAGGDGRQVAEPSTEAKNAQKGQPMAVGEETFRSLLEIFFFLSLHGDFKARLVTGKKGARVLRTLLQVANGAGKKGASSSLTRYLLLQSLCNIMRSREDRQRQRRRKGEVGSPLADVDDEQLQQLEELFKKLPEGAKPAANGEVDLGDKALATQLRDMLLDLNVVHAIAVNVCATPPPSSNVLCAAAQALKFLCEDSRHRGRAVQEGGIRTLLVAASGLEEFPDDQRNARQAAAQLCITTNPALFSYRESLDLVPCLAPLLKDRHELLQYEGALALTNLCALSEEVRMRAWLGGVWEGFEDLMFGENELLRAAGLEGWCNLSASPTVQTEIGKKMERFAAEKQEVQDMKLLLAFTRETNNPRAQSAAVAALAMLLANEKVARCLPAYSLFGNLALSLEEAKAEQEALIVRCVSALYNVWIELSSSEAGAETRMQIVKTLQRNQQKLTGDAQHLAKEVLTAELSQANTHTKESTPDSS.SEQ ID NO: 15 TGGT1_235470 (toxodb.org), Toxoplasma gondii GT1, CDS DNA, myosinA, TgMyoA heavy chainATGGCGAGCAAGACCACGTCTGAGGAGCTGAAAACGGCCACGGCGCTGAAGAAGAGGTCGTCCGATGTCCACGCGGTCGACCACTCCGGCAATGTGTACAAAGGATTTCAAATCTGGACGGACTTGGCGCCGTCGGTGAAGGAGGAGCCGGACCTGATGTTTGCCAAGTGCATCGTGCAGGCGGGGACAGACAAGGGGAACTTGACCTGCGTCCAGATCGATCCACCGGGCTTCGACGAACCGTTCGAAGTCCCGCAGGCGAATGCGTGGAACGTAAACAGCCTGATCGACCCCATGACGTACGGAGACATCGGCATGTTGCCTCACACGAACATTCCTTGCGTCCTCGACTTCCTCAAGGTGCGCTTCATGAAGAATCAAATCTACACGACTGCGGACCCGCTCGTCGTCGCCATCAATCCCTTCCGCGACCTCGGGAACACCACGCTCGACTGGATTGTTCGATACAGAGACACTTTCGACCTCTCCAAACTCGCGCCCCATGTTTTCTACACCGCCCGACGCGCGCTCGACAACCTCCACGCCGTCAACAAGTCGCAAACGATCATCGTGTCCGGTGAGTCTGGCGCGGGCAAGACGGAGGCGACGAAGCAGATTATGAGGTATTTTGCGGCGGCGAAGACGGGGTCGATGGATTTGCGGATTCAGAACGCGATCATGGCGGCGAATCCAGTGCTTGAGGCATTTGGAAATGCGAAGACGATTCGCAACAACAACTCGTCGCGTTTCGGACGCTTCATGCAGCTGGATGTGGGTCGCGAAGGAGGCATCAAGTTTGGCTCCGTCGTCGCCTTTCTCCTGGAAAAGTCGCGTGTTCTCACGCAGGACGAACAGGAGCGGTCGTACCACATCTTCTACCAAATGTGCAAGGGGGCGGACGCGGCGATGAAGGAGCGCTTCCATATCCTGCCGCTCTCGGAGTACAAGTACATCAATCCGTTGTGCCTGGACGCGCCAGGGATCGACGACGTCGCGGAGTTCCACGAAGTCTGCGAGTCGTTCCGGTCGATGAATCTGACGGAGGACGAAGTCGCGAGCGTGTGGAGCATCGTGAGTGGAGTGCTGCTGCTTGGCAACGTCGAGGTGACAGCGACGAAGGATGGGGGGATCGACGACGCCGCGGCGATCGAGGGGAAGAACTTGGAGGTTTTCAAAAAGGCCTGCGGGCTGCTCTTCCTCGACGCGGAGCGCATTCGCGAAGAGCTGACGGTGAAGGTTTCGTATGCGGGGAATCAGGAGATCCGCGGCCGGTGGAAGCAGGAAGACGGAGACATGCTCAAGTCGTCGCTCGCGAAGGCGATGTACGACAAGTTGTTCATGTGGATCATTGCCGTGTTGAACCGCAGCATCAAGCCTCCGGGCGGCTTCAAGATCTTCATGGGCATGCTCGACATCTTCGGCTTCGAAGTCTTCAAGAACAACTCGCTGGAGCAGTTCTTCATCAACATCACGAACGAAATGCTGCAGAAGAACTTCGTCGACATCGTCTTCGACCGCGAGAGCAAGCTGTATCGTGACGAGGGTGTCTCCTCCAAGGAGTTGATTTTCACCTCGAACGCAGAAGTGATCAAGATCTTGACGGCGAAGAACAACTCGGTGCTCGCTGCGCTCGAGGACCAGTGCCTCGCCCCTGGAGGCAGCGACGAAAAGTTCCTCTCGACCTGCAAGAACGCGCTGAAAGGAACCACCAAGTTCAAGCCTGCGAAGGTCTCTCCGAACATCAATTTCCTCATCTCGCACACTGTCGGCGACATCCAGTACAACGCCGAAGGCTTCCTCTTCAAAAACAAAGATGTCCTGCGAGCAGAAATCATGGAAATCGTGCAGCAAAGCAAGAACCCCGTCGTCGCGCAACTCTTCGCTGGCATCGTCATGGAGAAGGGGAAGATGGCCAAGGGACAACTGATTGGGTCGCAGTTCCTCTCGCAGCTGCAGAGCCTCATGGAACTTATCAACAGCACCGAGCCTCACTTCATTCGCTGCATCAAGCCGAACGACACGAAGAAGCCCCTCGACTGGGTGCCGTCGAAAATGCTCATTCAGCTGCACGCGCTCTCCGTCCTCGAGGCTCTTCAGCTCCGTCAACTCGGCTACTCTTACAGACGTCCGTTCAAGGAGTTCCTCTTCCAGTTCAAGTTTATCGACCTCTCGGCTTCTGAAAATCCAAATCTGGACCCCAAAGAAGCTGCGCTGAGACTCCTCAAAAGCAGCAAACTGCCCAGCGAAGAATACCAGCTCGGGAAGACAATGGTTTTCCTCAAGCAGACGGGCGCGAAAGAACTGACGCAGATTCAGAGAGAATGCCTTTCTTCTTGGGAGCCTCTCGTCTCAGTGCTCGAGGCGTACTACGCTGGCAGACGCCACAAGAAGCAGCTGCTGAAAAAGACCCCCTTCATCATTCGCGCCCAGGCTCACATCCGCAGACACCTGGTGGACAACAACGTCAGCCCCGCGACTGTTCAGCCGGCGTTCTAG.SEQ ID NO: 16 TGGT1_235470 (toxodb.org), Toxoplasma gondii GT1, amino acidsequence, myosin A, TgMyoA heavy chainMASKTTSEELKTATALKKRSSDVHAVDHSGNVYKGFQIWTDLAPSVKEEPDLMFAKCIVQAGTDKGNLTCVQIDPPGFDEPFEVPQANAWNVNSLIDPMTYGDIGMLPHTNIPCVLDFLKVRFMKNQIYTTADPLVVAINPFRDLGNTTLDWIVRYRDTFDLSKLAPHVFYTARRALDNLHAVNKSQTIIVSGESGAGKTEATKQIMRYFAAAKTGSMDLRIQNAIMAANPVLEAFGNAKTIRNNNSSRFGRFMQLDVGREGGIKFGSVVAFLLEKSRVLTQDEQERSYHIFYQMCKGADAAMKERFHILPLSEYKYINPLCLDAPGIDDVAEFHEVCESFRSMNLTEDEVASVWSIVSGVLLLGNVEVTATKDGGIDDAAAIEGKNLEVFKKACGLLFLDAERIREELTVKVSYAGNQEIRGRWKQEDGDMLKSSLAKAMYDKLFMWIIAVLNRSIKPPGGFKIFMGMLDIFGFEVFKNNSLEQFFINITNEMLQKNFVDIVFDRESKLYRDEGVSSKELIFTSNAEVIKILTAKNNSVLAALEDQCLAPGGSDEKFLSTCKNALKGTTKFKPAKVSPNINFLISHTVGDIQYNAEGFLFKNKDVLRAEIMEIVQQSKNPVVAQLFAGIVMEKGKMAKGQLIGSQFLSQLQSLMELINSTEPHFIRCIKPNDTKKPLDWVPSKMLIQLHALSVLEALQLRQLGYSYRRPFKEFLFQFKFIDLSASENPNLDPKEAALRLLKSSKLPSEEYQLGKTMVFLKQTGAKELTQIQRECLSSWEPLVSVLEAYYAGRRHKKQLLKKTPFIIRAQAHIRRHLVDNNVSPATVQPAF.SEQ ID NO: 17 TgMLC1 (ToxoDB TgGT1_257680, toxodb.org) CDS DNA, myosin lightchain MLC1atgagcaaggtcgagaagaaatgcccggtgtgctaccagaagctgccgaacccggcagatgttctgggtccgatggacaaggagttgaactatttcatgtggatgccaggcttcgagtggcgcccggaaccgaaggtgggggagtacgatggtgcctgtgagtcgccctcttgccgcgagggggggcgccctgcggcagacgaagacatgcaggaggctctcgaggagatggtggaggccgacgaaatgtatgcgcgcttcaacgcgagagcttccggaggaaaggtatccacgggagacgccatgattctcgcgcgccagctcggacttgccccgtcctacgcagacaaacaggcctttgaggaaaagagcggcgacaaccttgactacgccagcttccagaaattcgttggcaccagcacccaccccgaagacaacatcgaggacctcgtcgaagccttcgcatactttgacgtctctaagcacggttacctgacgcgcaagcagatggggaacatcctcatgacctacggagagcctctcaccacagaagagtttaatgccttggctgcggagtacttcacaagtgaccagatcgactacaggcaattctgcaaggcaatgctcgagcgaagggagtaa.SEQ ID NO: 18 TgMLC1 (ToxoDB TgGT1_257680, toxodb.org) amino acid sequence,myosin light chain MLC1MSKVEKKCPVCYQKLPNPADVLGPMDKELNYFMWMPGFEWRPEPKVGEYDGACESPSCREGGRPAADEDMQEALEEMVEADEMYARFNARASGGKVSTGDAMILARQLGLAPSYADKQAFEEKSGDNLDYASFQKFVGTSTHPEDNIEDLVEAFAYFDVSKHGYLTRKQMGNILMTYGEPLTTEEFNALAAEYFTSDQIDYRQFCKAMLERRE.SEQ ID NO: 19 TGGT1_269442, Toxoplasma gondii gt1, putative calmodulin, cds,length = 264atgtcaatggcgtggcctgattttgaggcgtggatgtcgaagaaactggcgtcctacaaccctgaggaggagttgatcaaatctttcaaggcttttgaccggtcgaacgacggcaccgtgtctgcggacgagctttctcaagttatgctcgctctcggcgagttgctttccgacgaagaagtcaaggccatgatcaaggaagccgacccgaacggcactggcaagatccagtacgccaactttgtcaagatgctgctgaaataa.SEQ ID NO: 20: TGGT1_269442, Toxoplasma gondii gt1, putative calmodulin,amino acid sequenceMSMAWPDFEAWMSKKLASYNPEEELIKSFKAFDRSNDGTVSADELSQVMLALGELLSDEEVKAMIKEADPNGTGKIQYANFVKMLLK. SEQ ID NO: 21 A biotin acceptor site amino acid sequenceSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIERS. SEQ ID NO: 22 A Myc tag amino acid sequenceEQKLISEEDL. SEQ ID NO: 23 A Tyl tag amino acid sequence EVHTNQDPLD.SEQ ID NO: 24Plasmodium falciparum motor domain amino acid sequence; PfMyoA heavy chainending at Lys 773MAVTNEEIKTASKIVRRVSNVEAFDKSGSVFKGYQIWTDISPTIENDPNIMFVKCVVQQGSKKEKLTVVQIDPPGTGTPYDIDPTHAWNCNSQVDPMSFGDIGLLNHTNIPCVLDFLKHRYLKNQIYTTAVPLIVAINPYKDLGNTTNEWIRRYRDTADHTKLPPHVFTCAREALSNLHGVNKSQTIIVSGESGAGKTEATKQIMRYFASSKSGNMDLRIQTAIMAANPVLEAFGNAKTIRNNNSSREGREMQLVISHEGGIRYGSVVAFLLEKSRIITQDDNERSYHIFYQFLKGANSTMKSKFGLKGVTEYKLLNPNSTEVSGVDDVKDFEEVIESLKNMELSESDIEVIFSIVAGILTLGNVRLIEKQEAGLSDAAAIMDEDMGVFNKACELMYLDPELIKREILIKVTVAGGTKIEGRWNKNDAEVLKSSLCKAMYEKLFLWIIRHLNSRIEPEGGEKTFMGMLDIFGFEVEKNNSLEQLFINITNEMLQKNFVDIVFERESKLYKDEGISTAELKYTSNKEVINVLCEKGKSVLSYLEDQCLAPGGTDEKFVSSCATNLKENNKFTPAKVASNKNFIIQHTIGPIQYCAESFLLKNKDVLRGDLVEVIKDSPNPIVQQLFEGQVIEKGKIAKGSLIGSQFLNQLTSLMNLINSTEPHFIRCIKPNENKKPLEWCEPKILIQLHALSILEALVLRQLGYSYRRTFEEFLYQYKFVDIAAAEDSSVENQNKCVNILKLSGLSESMYKIGKSMVFLKQEGAKILTKIQREK. SEQ ID NO: 25Toxoplasma gondii motor domain amino acid sequence; TgMyoA heavy chainending at Cys 775MASKTTSEELKTATALKKRSSDVHAVDHSGNVYKGFQIWTDLAPSVKEEPDLMFAKCIVQAGTDKGNLTCVQIDPPGFDEPFEVPQANAWNVNSLIDPMTYGDIGMLPHTNIPCVLDFLKVRFMKNQIYTTADPLVVAINPFRDLGNTTLDWIVRYRDTFDLSKLAPHVEYTARRALDNLHAVNKSQTIIVSGESGAGKTEATKQIMRYFAAAKTGSMDLRIQNAIMAANPVLEAFGNAKTIRNNNSSREGREMQLDVGREGGIKEGSVVAELLEKSRVLTQDEQERSYHIFYQMCKGADAAMKERFHILPLSEYKYINPLCLDAPGIDDVAEFHEVCESFRSMNLTEDEVASVWSIVSGVLLLGNVEVTATKDGGIDDAAAIEGKNLEVEKKACGLLELDAERIREELTVKVSYAGNQEIRGRWKQEDGDMLKSSLAKAMYDKLFMWIIAVLNRSIKPPGGFKIFMGMLDIFGFEVEKNNSLEQFFINITNEMLQKNEVDIVEDRESKLYRDEGVSSKELIFTSNAEVIKILTAKNNSVLAALEDQCLAPGGSDEKFLSTCKNALKGTTKFKPAKVSPNINFLISHTVGDIQYNAEGFLEKNKDVLRAEIMEIVQQSKNPVVAQLFAGIVMEKGKMAKGQLIGSQFLSQLQSLMELINSTEPHFIRCIKPNDTKKPLDWVPSKMLIQLHALSVLEALQLRQLGYSYRRPFKEFLFQFKFIDLSASENPNLDPKEAALRLLKSSKLPSEEYQLGKTMVFLKQTGAKELTQIQREC. SEQ ID NO: 26Plasmodium falciparum motor domain including the ELC binding site, aminoacid sequence, PfMyoA heavy chain ending at Asn798MAVTNEEIKTASKIVRRVSNVEAFDKSGSVFKGYQIWTDISPTIENDPNIMFVKCVVQQGSKKEKLTVVQIDPPGTGTPYDIDPTHAWNCNSQVDPMSFGDIGLLNHTNIPCVLDFLKHRYLKNQIYTTAVPLIVAINPYKDLGNTTNEWIRRYRDTADHTKLPPHVFTCAREALSNLHGVNKSQTIIVSGESGAGKTEATKQIMRYFASSKSGNMDLRIQTAIMAANPVLEAFGNAKTIRNNNSSREGREMQLVISHEGGIRYGSVVAFLLEKSRIITQDDNERSYHIFYQFLKGANSTMKSKFGLKGVTEYKLLNPNSTEVSGVDDVKDFEEVIESLKNMELSESDIEVIFSIVAGILTLGNVRLIEKQEAGLSDAAAIMDEDMGVFNKACELMYLDPELIKREILIKVTVAGGTKIEGRWNKNDAEVLKSSLCKAMYEKLFLWIIRHLNSRIEPEGGEKTFMGMLDIFGFEVEKNNSLEQLFINITNEMLQKNFVDIVFERESKLYKDEGISTAELKYTSNKEVINVLCEKGKSVLSYLEDQCLAPGGTDEKFVSSCATNLKENNKFTPAKVASNKNFIIQHTIGPIQYCAESFLLKNKDVLRGDLVEVIKDSPNPIVQQLFEGQVIEKGKIAKGSLIGSQFLNQLTSLMNLINSTEPHFIRCIKPNENKKPLEWCEPKILIQLHALSILEALVLRQLGYSYRRTFEEFLYQYKFVDIAAAEDSSVENQNKCVNILKLSGLSESMYKIGKSMVFLKQEGAKILTKIQREKLVEWENCVSVIEAAILKHKYKQKVN.SEQ ID NO: 27Toxoplasma gondii motor domain including the ELC binding site, amino acidsequence, TgMyoA heavy chain ending at Leu 800MASKTTSEELKTATALKKRSSDVHAVDHSGNVYKGFQIWTDLAPSVKEEPDLMFAKCIVQAGTDKGNLTCVQIDPPGFDEPFEVPQANAWNVNSLIDPMTYGDIGMLPHTNIPCVLDFLKVRFMKNQIYTTADPLVVAINPFRDLGNTTLDWIVRYRDTFDLSKLAPHVFYTARRALDNLHAVNKSQTIIVSGESGAGKTEATKQIMRYFAAAKTGSMDLRIQNAIMAANPVLEAFGNAKTIRNNNSSRFGRFMQLDVGREGGIKFGSVVAFLLEKSRVLTQDEQERSYHIFYQMCKGADAAMKERFHILPLSEYKYINPLCLDAPGIDDVAEFHEVCESFRSMNLTEDEVASVWSIVSGVLLLGNVEVTATKDGGIDDAAAIEGKNLEVFKKACGLLFLDAERIREELTVKVSYAGNQEIRGRWKQEDGDMLKSSLAKAMYDKLFMWIIAVLNRSIKPPGGFKIFMGMLDIFGFEVFKNNSLEQFFINITNEMLQKNFVDIVFDRESKLYRDEGVSSKELIFTSNAEVIKILTAKNNSVLAALEDQCLAPGGSDEKFLSTCKNALKGTTKFKPAKVSPNINFLISHTVGDIQYNAEGFLFKNKDVLRAEIMEIVQQSKNPVVAQLFAGIVMEKGKMAKGQLIGSQFLSQLQSLMELINSTEPHFIRCIKPNDTKKPLDWVPSKMLIQLHALSVLEALQLRQLGYSYRRPFKEFLFQFKFIDLSASENPNLDPKEAALRLLKSSKLPSEEYQLGKTMVFLKQTGAKELTQIQRECLSSWEPLVSVLEAYYAGRRHKKQLL.SEQ ID NO: 28C. elegans Unc45b amino acid sequence (Genbank Accession No. AAD01976.1)mvarvqtaee irdegnaavk dqdyikadel ytealqlttd edkalrpvly rnramarlkrddfegaqsdc tkalefdgad vkalfrrsla reqlgnvgpa fqdakealrl spndkgivevlqrlvkannd kikqttslan kvtdmeklaf rgeakdteqk mtalnnllvl cresesgatgvwnqgalvpf vinlindase neevtvtair ildetiknsv rcmkflamhd pdgpksvrfvcrlmckkstk dfvdatgilv qrvfnamakm drqkemkpdp evaeankiwi irvllelqemlqdpkvgavq retcidlflk nlmhmdggip rgwswkfvee rgllalldva sqipelceypvsaetrqhva iclqrleedm vfdtkrtifk ekvdmffnal isrctnddeg hkyriklscflitmlqgpvd iginlitndq ltpimlemaa sqdhlmqgia aelivatvsk herainmlkvgipvlralyd sedptvkvra lvglckigaa ggddiskatm keeavislak tckkflletekysvdirrya ceglsylsld advkewivdd slllkalvll akkagalcvy tlatiyanlsnafekpkvde emvklaqfak hhvpethpkd teeyvekrvr alveegavpa cvaysktesknaleliarsl lafaeyedlr griiaeggtv lclrltkeas gegkikagha iaklgakadpmisfpgqray evvkplcdll hpdvegkany dslltltnla sysdsirgri lkekaipkieefwfmtdheh lraaaaelll nllffekfye etvapgtdrl klwvlysaev eeerlsrasaagfailtede nacarimdei kswpevfkdi amhedaetqr rglmgianim hssnklcseivssevfrvlv avtklgtinq eragsteqak rgleaaekfg likatdreiy erenqmstiq e.SEQ ID NO: 29Podospora anserina CRO1 amino acid sequence (Genbank Accession No. CAA76144.1)matvaeaaaa apeplgrldq tllifaglme ggkedeetvr elgeltrlln ddvevtkkgetsvttvidsd cvdtilcyld mrqpdvvrah aalctsaylk aagedggkkl aeffhdrvrrgtyddyivaf cvaatifpiv pdltselfls egflaslgpl mrrkwksrkv etacleminaacmnsacrea vqkyctewle eiveqdpdda vksmhtvdpd mhlqegsism rrhslqvqnlaavvlaklra vpstaatagp eariqpatts iedlskrftr mlldedeieh vqpsieglayaslqpkvkes lskdsktlkr lvkaldeapp rspmiygals iftnitryrp ietdeekrirqlkayanaag klqqvdpine dehvterckr vfeagltpvl ikqsksgsaa slaliisiihalstppplrg qlaqqgavrl liaawtalpe tengpkraaa qalarilist npalvfggtrpipqsaairp lasiltpdpt adrrdllptf eslmaltnla stdddtrksi irtawddveeqlfnpnsrvc taavelvcnl vqdpeqtlal fgdgspkakn rvkvivalad aedpktrsaaggalasltgf devvravmgl ergvevvlgl crderedlrh rgavvvrnmv fsegevgrlargklveggav ealmecakgs krrevvevvv qaaeglmgeg gk. SEQ ID NO: 30S. cerevisiae She4p amino acid sequence (Genbank Accession No. DAA10818.1)mplcekgndp idsstidslc aafdktlkst pdvqkyndai ntifqlrqks esgkmpadltnsealkdrqk ieeiltrsyq dhsesrvhls kliqndipfa lnlfeilsrs sihvfvgcfsnkdatialln elqirihyge dthvtyllsi ilqllnkfky nfkevrflvk elilrisedevksmmliifa elqssfqkdf dkavvdfmss liveaeidvg ndplsiivkt lselypslttlcseifltkg lsklfkkrvf eeqdlqftke llrllssaci detmrtyite nylqllerslnvedvqiysa lvlvktwsft kltcinlkql seifinaisr rimpkienvn esavkleevpkvemsveala ylslkasvki mirsnesfte illtmiksqk mthclygllv imanlstlpeesngssqsin dlknyadlkg pgadkvgaek eskedillfn ekyilrteli sflkremhnlspnckqqvvr viynitrskn fipqcisqgg ttiileylan kqdigepiri lgcraltrmliftnpglifk kysalnaipf lfellprstp vddnplhnde qikltdnyea llaltnlassetsdgeevck hivstkvyws tienlmlden vplqrstlel isnmmshplt iaakffnlenpqslrnfnil vkllqlsdve sqravaaifa niattiplia kelltkkeli enaiqvfadqiddielrqrl lmlffglfev ipdngtnevy pllqenqklk dalnmslkrg dsgpefsaaipvilakikv. SEQ ID NO: 31Drosophila melanogaster UNC-45 amino acid sequence (DmUNC) (GenbankAccession No. AAK93568)mtntinseev sdagsykdkg neafkasrwe eavehygkai kagskhkela vfyknraaaylklgkyenav edcteslkaa pgdpkalfrr aqayealekf eeaykdatal fkadpgnktvqpmlqrlhvv veersarnak tstkvkqmmd ltfdlatpid krraaannlv vlakeqtgaellykdhciak vasltkvekd qdiyvnmvhl vaalcensve rtkgvltelg vpwfmrvldqkhencvstaq fclqtilnal sglknkpdsk pdkelctrnn reidtlltcl vysitdrtisgaardgviel itrnvhytal ewaerlveir glcrlldvcs eledykyesa mditgssstiasvclariye nmyydeakar ftdqideyik dkllapdmes kvrvtvaita llngpldvgnqvvaregilq milamattdd elqqrvacec liaasskkdk akalceqgvd ilkrlyhskndgirvralvg lcklgsyggq daairpfgdg aalklaeacr rflikpgkdk dirrwaadglayltldaeck ekliedkasi halmdlargg nqsclygvvt tfvnlcnaye kqemlpemielakfakqhip eehelddvdf inkritvlan egittalcal akteshnsqe liarvinavcglkelrgkvv qeggvkallr malegtekgk rhatqalari gitinpevsf sgqrsldvirpllnllqqdc talenfeslm altnlasmne svrqriikeq gvskieyylm edhlyltraaaqclonlvms edvikmfegn ndrvkflall cededeetat acagalaiit sysvkccekilaiaswldil htlianpspa vqhrgiviil nminageeia kklfetdime llsglgqlpddtrakareva tqclaaaery riiersdnae ipdvfaensk iseiidd. SEQ ID NO: 42Truncated PfLINC lacking N-terminal TPR domain (Genbank Accession No.XP_001348369.1 beginning at N178)NKDNNNKSEINRINEKLRDIMKIIDQNKNDPYKNISNIKKYLLDENTNNINDIEENNKKIKLLHSIYNQKFYILLKENIFLFLFDFIKKNNDISNYDDCNNNNNNNNLYSHNINSLLLEKTAIYVIYKILSKLDNEHIIIENSKDDNYNKNRNICINKLDNSKLQYYYDLDYIKMILSFNEYFTKDWIYNYIKKKINILENLKFSKDETLYKEHVDILIYIINIMKYVYVINNDYILNIINSYYLNSDNSNINNSGINALTFLCKKKQFLTQNSKDNRKKKNDLLEMLKKENYLNIQNNIQNNNNNNNYYFYKNEFIEFHFSDSHKYPLCINSEIKKIIQNVIGMYEHFSSSIEYTLILIFTLLHDPQRPKEKDIEMNDVIYDCIDNYFHHNENILIEWFVCIKCLFLVDKNIILNYLIGKTEYIVKILHFITNCIGRKTKEELSIYIDVLLLLLNISEIRFMFTNYIDMYINIMKSLNYDQCFLKLLLGTFKLYMHNIDFKQQIQDNVDLFFYAKEILKQFLLTYDNDADGKNNTNNDKREENEEQTSHLNYSNLNSYTCCVKKCDDNDTKKKDIINQKKDEKNKKHCELVDKKKKDHTYIHSNMSCEKTLKDLIEMLFYLSLHIEFKKQLLEEKNNYILFFLIKVGHDINKKKLDNTYKYIYCNTINNLILTKNDEKIKRREINKTNLSNFDNEQIEALEQFYDKLPKEARPKTDPLYDYGDEETSNKLIDLLLYNEKYQMNHINDKNKNNNNNNNINNGNVSPLSSKCSYTNGTIINIIYNFINSNFFTTNIAESVCEIISKFVKNTNNIGIVLVNNGLKTLLLASKHITNKKNCALALSEIFIYTNPKLIHFYEAYDSLPLLIEQLKSDEELLIFKTLMAITNILTIDENVAIKAMQLNLWYKCFDILSTENEYIKSASLECICNLCSQSHVHQYIYDKYQTIMKSKNESDKDILFVDIQIIYSFTMEYQNYKCVFAATGALGMLSSDLRLPYYLVRTKGIDHIFSSFNNTTDQNILLRILTFFNNIMTCDDIPDDILKKIKTYVEKKKDLNEENTQMANFILQ. SEQ ID NO: 43PfGAP 45 Plasmodium falciparum GAP45 Sequence, CDS, Genbank Accession No.XM_001350588.1atgggaaataaatgttcaagaagcaaagtaaaggaacccaaacgtaaagatattgatgaattagctgaacgtgaaaatttaaaaaaacaatctgaagaaataattgaagaaaaaccagaagaagttgttgagcaagtagaagaaacacatgaagaacctcttgaacaagaacaggaactggatgaacagaaaatagaagaagaagaagaagaacctgaacaagtaccaaaagaagaaatagattatgcaactcaagaaaataaatcatttgaagaaaaacatttagaagatttagaaagatctaattcagatatttattcagaatctcaaaaatttgataatgctagtgataaattagaaacaggaactcaattaaccttatctactgaagccactggtgccgtacaacaaataactaaattaagtgaacccgcccatgaagaaagtatatattttacttatagatctgtaacaccttgtgatatgaataaactcgatgaaaccgctaaagttttttcaagaagatgtggatgtgatcttggtgaacgtcatgatgaaaatgcatgtaaaatttgtagaaaaattgatttatccgatacacctttattgagctaa. SEQ ID NO: 44PfGAP 45 Plasmodium falciparum GAP45 amino acid sequence; GenbankAccession No. XP_001350624.1MGNKCSRSKVKEPKRKDIDELAERENLKKQSEEIIEEKPEEVVEQVEETHEEPLEQEQELDEQKIEEEEEEPEQVPKEEIDYATQENKSFEEKHLEDLERSNSDIYSESQKFDNASDKLETGTQLTLSTEATGAVQQITKLSEPAHEESIYFTYRSVTPCDMNKLDETAKVFSRRCGCDLGERHDENACKICRKIDLSDTPLLS.SEQ ID NO: 45TgGAP 45 Toxoplasma gondii GAP45 Sequence, CDS, Genbank Accession No.XM_002366039.1gtgagatttcctttcagcgaattttgtgatctcaagagcgagaggcacaactgtatttgagaacttttgctttttcagcgttgcacgagttttaaatctcggtgtgactgttcgaaaccctgtcatttccctccgagaagatcggtcgacggcgcgcggacaaccgcgtgcgtaaaaggtgtttgctatttcgccggggagaaaagcggccgtaaagtcggaagacttgccagacgagacgagaaactgggtgcggcacggcatttcatcattctgtatgtgttgttttccattcgaagaaagttttgtttctcgcggcagagggaaaggcgcgcaccgaacctgcccgcagtttccggtacatccacgcacacacttcggtggctgaaacgccgcgattgttccgttcttgtgttcgcgacttcgactctctgaatcgctcccccacacatcttttgttgtcgccccctcaactttttcgcactttttcgattcgaaatgggaaacgcgtgcaagaagaacacggccaagacgccgacccggaaggaggcggaggacctggctgagaaggagcggcaggagcgggaggcgaaggagaaggctgaggctgaagagaaggctcgcgccgaagcggagaagaacgcagcagacaaagcggaggctgaacgtagagcggcagaggcgcgagagcgcgaagaatcagccaggaaggaggcggaggctgaggcggcccgcaaggccgaagcagaggcggctgaggccgagcgcctccggaaggaagcagagaagaaaaaggcggaggaggcgaaacgcaaggcggaggaggagcagcgcgccgcagccgaggaagcggagcaaagagcgcgggaggaggccgagcgccgcaaagctgaagctgcggcggcagcggagcgcgagcgccagatgcaggaggcgctgaagcaagaggaaatgtcaccgagagagaagtacgacaagttagccagccccgaagactccgcatccgagaccacgatggcgacacagccgcagaaagtcgccgagcacagcagcgcggcggtcacagacagatcagtggtggggtacaccgtgactccatgcgacatggcatcaattgacgagacagctaagtacttgtcaaagcgctgcggttgcgacctaggcgaccaacacgacgaaaacgagtgccctatttgccgccacatcgacttgtcggatgcacccttgttgaactgagtgcgtaactgtttctgtgtttttctactctacggccccggcttcgggatctgtgtctgtatagcgtgcgcttataaacgcgtaaacttcgtgtttaagaaagattagcaagggttaggacggtgaagaagagtttgggaccgtgttttttcgacaaacgtcgggttgctagtatcgcacgtaggtgtgcactaccccgctctccatgtaggcgtagtctgtgtagcaacaggacggttggcggcaaacagaaagggaggaaatttctgcgggttcttcgagtctgagctgtcgcgaaaagctccaaaagcgaactggctccccgcagtctatgggggacgcacccgccggtaggggtgaagtggctcacaggcttcgcccgcctgtccgtacgtgcagggcagaatcctgcccgaacgtgagaacaaccgtcattccccgtcgtcacatagttctttttctcgaccatccccacgtgacaccactggtgtgcgaatgcggcgcagcgttcgactgtcttccgcgaaaggcgatgcttacaaacgcacgctcgcatgacatacgtgccgtaaagaggaaacccttet. SEQ ID NO: 46TgGAP 45 Toxoplasma gondii GAP45 amino acid sequence, Genbank Accession No.XP_002366080.1MGNACKKNTAKTPTRKEAEDLAEKERQEREAKEKAEAEEKARAEAEKNAADKAEAERRAAEAREREESARKEAEAEAARKAEAEAAEAERLRKEAEKKKAEEAKRKAEEEQRAAAEEAEQRAREEAERRKAEAAAAAERERQMQEALKQEEMSPREKYDKLASPEDSASETTMATQPQKVAEHSSAAVTDRSVVGYTVTPCDMASIDETAKYLSKRCGCDLGDQHDENECPICRHIDLSDAPLLN. SEQ ID NO: 47Toxoplasma gondii ELC1 nucleotide sequence, also referred to herein asTgELC1 nucleotide sequence (ToxoDB v7.3 TgGT1_107770)Atgacctgccctccccgcgtccgtgaggccttcgccctcttcgacactgacggagatggtgagatctctggccgcgacctcgtcctcgccatccgctcatgcggtgtgtctcccaccccagacgaaatcaaggcactccccatgtcaatggcgtggcctgattttgaggcgtggatgtcgaagaaactggcgtcctacaaccctgaggaggagttgatcaaatctttcaaggcttttgaccggtcgaacgacggcaccgtgtctgcggacgagctttctcaagttatgctcgctctcggcgagttgctttccgacgaagaagtcaaggccatgatcaaggaagccgacccgaacggcactggcaagatccagtacgccaactttgtcaagatgctgctgaaataaggatccattgt. SEQ ID NO: 48Toxoplasma gondii ELC1 amino acid sequence, also referred to herein as TgELC1MTCPPRVREAFALFDTDGDGEISGRDLVLAIRSCGVSPTPDEIKALPMSMAWPDFEAWMSKKLASYNPEEELIKSFKAFDRSNDGTVSADELSQVMLALGELLSDEEVKAMIKEADPNGTGKIQYANFVKMLLK.SEQ ID NO: 49: Plasmodium falciparum 3D7 myosin light chain, (mayalso be referred to herein as an “essential type light chain”, an“essential light chain”, a PfELC, and/or an ELC1) putative (PFF1320c) mRNA,Genbank Accession No. XP_966255.2:MEEIINEVELTSLFNKISEGSRTIHFEDAMEIIYKMGYVPSKEDINEFNNMTKGVCSLSNIKKFCNKIRSLNYSTEGLLDIFHFYDTNKTGKISKEKLKLLFTTVGSKMSVDEMDTIINELCNNDENIDYKEFLNRILN.SEQ ID NO: 50: Plasmodium falciparum 3D7 myosin light chain (may also bereferred to herein as an “essential type light chain”, an “essential lightchain”, a PfELC, and/or an ELC1), putative (PFF1320c) mRNA,NCBI Reference Sequence: XM_961162.2:atggaggaaa taattaatga agtggaattg acttctcttt ttaacaagat atcagaaggg tcgagaactatccattttga ggatgccatg gaaataatat ataaaatggg ttatgttcct tcaaaagaag atataaatgaatttaacaac atgacaaaag gtgtttgttc tttatccaat ataaaaaagt tctgcaataa aataaggtcattgaattatt ccactgaagg tttgttggat atatttcatt tttatgatac aaataaaaca ggaaaaatttctaaagaaaa acttaaactc ttatttacaa cagttggttc aaaaatgtcg gttgatgaaa tggatacaataataaatgaa ttatgtaata acgatgaaaa catagactat aaggaatttc taaacaggat attaaattag.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide schematic diagrams of T. gondii myosin motorcomplex and subunit structure of TgMyoA. FIG. 1A shows TgMyoA (i.e.,TgMyoA heavy chain with its bound light chains, TgMLC1 and TgELC1) andTgGAP45 are anchored to the inner membrane complex (IMC) viatransmembrane protein TgGAP50. This multiprotein complex is referred toas the myosin motor complex. Short actin filaments are located betweenthe parasite plasma membrane and the IMC, and are thought to beconnected to ligands on the host cell surface through linker protein(s)that bind to the cytosolic tails of surface adhesins. TgMyoA (attachedto the IMC) displaces the actin filaments (attached to the substrate)causing the parasite to move relative to the substrate. Note thatalternative models of parasite motility are emerging (47, 48) in whichTgMyoA plays a different but still important role in generating theforce required for motility. Figure adapted from (12). FIG. 1B shows thesubunit structure of TgMyoA. The heavy chain motor domain contains theactin and ATP binding sites, followed by the lever arm, to which thelight chains TgMLC1 and TgELC1 are believed to bind. Rotation of theTgMyoA lever arm produces a power stroke that moves actin filaments andpropels the parasite forward.

FIGS. 2A and 2B show schematics of the expressed proteins. FIG. 2A showsa TgMyoA heavy chain construct that contains the motor domain (red) andthe light chain-binding region (black), followed by a Bio-tag and aFLAG-tag. FIG. 2B shows that TgUNC consists of three domains: TPR (atleft end), central (middle section) and the UCS region (at right end),followed by a Myc tag. The three different TgUNC constructs used duringcoexpression with TgMyoA heavy chain are shown below the schematic.

FIG. 3 shows a Western blot of results demonstrating co-expression ofTgMyoA with the chaperone TgUNC in Sf9 cells produces soluble heavychain. Sf9 cells were co-infected with recombinant baculovirus codingfor the TgMyoA heavy chain (HC) and its light chain TgMLC1. The Westernblot shows the total (T) and soluble (S) protein fractions after 72 hinfection in the absence (left two lanes) or presence (right two lanes)of co-expressed TgUNC. TgUNC was detected using anti-Myc antibody, whileTgMyoA heavy chain was detected with anti-FLAG antibody.

FIGS. 4A and 4B provide images of an SDS-gel showing characterization ofpurified TgMyoA and a graph. FIG. 4A shows results of SDS-gel analysisof purified motor proteins. Lane 1, purified protein resulting fromco-expression of TgMyoA heavy chain (HC), TgMLC1 light chain, and TgUNC.TgUNC only binds to unfolded protein and does not co-purify with TgMyoA.Lane 2, molecular weight standards. Lane 3, purified protein resultingfrom coexpression of TgMyoA heavy chain, TgMLC1 and TgELC1 light chains,and TgUNC. FIG. 4B shows a graph of results demonstrating sedimentationvelocity of TgMyoA heavy chain expressed with TgMLC1. A sedimentationcoefficient of 7.7S was determined by curve-fitting to one species. Thesymmetrical nature of the boundary indicates that a homogeneous speciesis present. OD, optical density.

FIG. 5 shows a graph indicating that the number of bound light chainsdetermines the speed of actin movement in an in vitro motility assay.The Gaussian fit of speeds for TgMyoA with TgMLC1 only was 1.5±0.2 μm/s(mean±SD, n=2,341 filaments) (solid triangles), compared to 3.4±0.7 μm/s(mean±SD, n=4,774 filaments) for TgMyoA with both light chains (solidcircles). Conditions: 25 mM imidazole, pH 7.5, 50 mM KCl, 1 mM EGTA, 4mM MgCl₂, 10 mM DTT, 5 mM MgATP, 0.5-0.7% (w/v) methylcellulose, andoxygen scavengers, 30° C. Addition of 1.2 mM calcium (i.e., 0.2 mM freecalcium) did not affect motility speed (open symbols). In the presenceof calcium, TgMyoA with TgMLC1 only moved actin at 1.5±0.2 μm/s(mean±SD, n=619 filaments), and TgMyoA containing both TgMLC1 and TgELC1moved actin at 3.3±0.6 μm/s (mean±SD, n=1,729 filaments). The valuesobtained in calcium versus EGTA were indistinguishable (p>0.1,Kolmogorov-Smirnov test). For each dataset, the bin with the highestspeed was normalized to one for presentation purposes. Data wereobtained from three protein preparations of TgMyoA with both lightchains, and two protein preparations of TgMyoA with TgMLC1 only.

FIG. 6 shows immunoblot results demonstrating that both TgMLC1 andTgELC1 are bound to the same heavy chain. TgMyoA heavy chain (HC) wasco-expressed in Sf9 cells with both 3×Myc-MLC1 and 3×HA-ELC1. TgMyoA wasco-immunoprecipitated from cell lysates using either anti-HA antibody(to immunoprecipitate TgELC1; left panels), or anti-TgMLC1 antibody(right panels), confirming that each of the light chains bind to TgMyoAheavy chain. Furthermore, TgMLC1 is present in the TgELC1 pulldowns andvice versa, demonstrating that the two light chains are simultaneouslypresent on the same MyoA heavy chain. TgUNC (˜126 kDa) was detected withanti-Myc antibody, TgMyoA heavy chain (˜104 kDa) with anti-FLAG,3×Myc-TgMLC1 (˜29 kDa) with anti-Myc on left panels, anti TgMLC1 onright panels and 3×HA-ELC1 (˜18 kDa) with anti-HA.

FIGS. 7A and 7B provide graphs of results of in vitro motility andsteady-state actin-activated ATPase assays. FIG. 7A shows in vitromotility speed as a function of MgATP concentration. Solid line is a fitto Michaelis-Menten kinetics. Vmax=4.6±0.3 μm/s and KM=1.3±0.3 mM MgATP.FIG. 7B shows steady-state actin-activated ATPase assay as a function ofskeletal actin concentration. Data were fit to the Michaelis-Mentenequation: Vmax=84±9.5 s−1 and Km=136±22 04. Conditions: 10 mM imidazole,pH 7.0, 5 mM NaCl, 1 mM MgCl₂, 1 mM NaN₃, 5 mM MgATP and 1 mM DTT at 30°C.

FIG. 8 shows Western blot results demonstrating that the TPR domain ofTgUNC is not required for solubility of TgMyoA. Sf9 cell infections wereperformed with three TgUNC constructs (see FIG. 2B). Expression andsolubility of TgMyoA were determined by Western blotting. Total (T) andsoluble (S) fractions were probed with either anti-FLAG (top panel) forTgMyoA heavy chain (HC) or anti-Myc (bottom panel) for TgUNC and itstruncations.

FIG. 9 provides a graph illustrating in vitro motility speeds ofpurified TgMyoA expressed with various TgUNC constructs. When TgMyoA wasexpressed with either full-length TgUNC or ΔTPR, the sliding speed withskeletal actin was similar: full-length TgUNC (3.4±0.7 μm/s, mean±SD,n=4,774 filaments) and ΔTPR (3.1±0.5 μm/s, mean±SD, n=2,048 filaments).Speed decreased when TgMyoA was expressed in the presence of the UCSdomain only (0.7±0.2 μm/s, mean±SD, n=1,296 filaments). The bin with thehighest speed was normalized to one for presentation purposes. Data wereobtained from three preparations of TgMyoA co-expressed with TgUNC, twoprotein preparations of TgMyoA co-expressed with ΔTPR, and one proteinpreparation of TgMyoA co-expressed with the UCS domain.

FIG. 10 shows a multiple sequence alignment of TgUNC (ToxoDBTgGT1_(—)249480; GenBank Accession Number EPR63428.1, SEQ ID NO:12) withthe three canonical members of the UCS family of myosin chaperones,i.e., Caenorhabditis elegans Unc45b [(AAD01976.1); SEQ ID N0:28],Podospora anserina CRO1 [(CAA76144.1); SEQ ID N0:29] and Saccharomycescerevisiae She4p [(DAA10818.1); SEQ ID N0:30]. The alignment wasgenerated using Multalin (http://multalin.toulouse.inra.fr/multalin/)(49) and processed using BoxShade v3.31C(http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::boxshade). Identicalresidues are highlighted in black, similar residues in grey, and thestart of the three domains (TPR domain, Central domain, and UCS domain)are shown above the sequence alignment. In TgUNC, L161 marks the startof the central domain, and E682 the start of the UCS domain. The percentidentity/percent similarity of full-length TgUNC with the other UCSfamily members are: Unc45b, 17.4/28.7; CRO1, 9.5/16.3; She4p, 9.3/17.4.Comparing only the UCS domains, the percent identity/percent similaritywith TgUNC is: Unc45b, 20.4/31.1; CRO1, 17.4/27.3; She4p, 15.3%/25.3.

FIG. 11 shows an amino acid sequence comparison of ARM motif residues inDrosophila UNC-45 and TgUNC. Sequence alignment of Drosophilamelanogaster UNC-45 (DmUNC) [(accession number AAK93568); SEQ ID N0:31]with TgUNC [(ToxoDB TgGT1_(—)249480; GenBank Accession NumberEPR63428.1); SEQ ID NO:12], using the program ALIGN(http://xylian.igh.cnrs.fr/bin/align-guess.cgi) (50), shows 22.5%identity. The crystal structure of DmUNC has been determined (PDB ID:3NOW) (51). Amino acid positions that conform to the ARM repeatconsensus sequence and structural requirements proposed by Andrade etal. (52) were previously identified by Lee et al. (51), and arehighlighted here. 40.4% of these ARM consensus residues (63 of 156) areidentical in TgUNC (also highlighted). The last five ARM motifs (17-21,motif 17 starts around Arg715) are the proposed sites of interactionwith myosin (51).

DETAILED DESCRIPTION

The invention, in part, relates to methods of preparing functional classXIV myosin and use of the prepared class XIV myosin in assays such asfunctional assays, drug screening assays, etc. In addition, theinvention in some embodiments includes methods of preparing and usingclass XIV myosin, which may in some embodiments be associated with oneor more myosin light chains. It has now been discovered thatco-expression of a class XIV heavy chain polypeptide-encodingpolynucleotide expressed with a parasite co-chaperonepolypeptide-encoding polynucleotide can be used to prepare a functionalclass XIV myosin.

Myosins make up a family of ATP-dependent motor proteins and areinvolved in a number of motility processes. Motor proteins are a classof molecular motors that are able to move along the surface of asuitable substrate. Motor proteins are powered by the hydrolysis of ATPand are responsible for actin-based motility. The myosin genes are partof a large superfamily of genes whose protein products share the basicproperties of actin binding, ATP hydrolysis (ATPase enzyme activity),and force transduction. Virtually all eukaryotic cells contain myosinisoforms, and the structure and function of myosin is strongly conservedacross species. Most myosin molecules are include three domains, (1) thehead domain that binds actin and uses ATP hydrolysis to generate forceto move the actin, (2) the neck domain that is a binding region for oneor more myosin light polypeptide chains and can also act as a “lever” totransduce force generated by the catalytic motor domain, and (3) thetail domain. Class XIV myosins are a myosin group that is found in theApicomplexa phylum, including, importance, including Toxoplasma gondii(toxoplasmosis) and Plasmodium spp. (malaria). TgMyoA, the class XIVmyosin in T. gondii, is necessary for efficient parasite motility, forinvasion into and egress from host cells, and for virulence in a mousemodel of infection. The speed of actin movement by myosin depends onboth the kinetics of nucleotide binding and release from the motordomain, as well as the length of the lever arm, which is determined bythe number of bound light chains.

It has now been discovered that expression of functional TgMyoA in aheterologous system requires co-expression with a co-chaperone of theUCS family. In some embodiments of the invention the co-chaperone is aT. gondii co-chaperone. The motor needs to bind two light chains (TgMLC1and TgELC1) to propel actin at fast speeds. Heterologous expression ofthis unique myosin is the only way to obtain sufficient purified proteinfor structure-function studies as well as drug testing but not limitedto Aplicomplexa members Toxoplasma gondii and Plasmodium falciparum.

Expression of Class XIV Myosin

A strategy of co-expression of a co-chaperone polypeptide with a classXIV heavy chain has now been used to prepare and isolate functionalTgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, inexpression system cells. Functional TgMyoA, also referred to herein as a“motor protein” or a “motor complex”, is required for the parasite toefficiently move and invade host cells. The T. gondii genome containsone myosin co-chaperone of the UNC-45/Cro1/She4p (UCS) family, which isreferred to herein as TgUNC. Functional protein was obtained when theTgMyoA heavy and light chain(s) were co-expressed with TgUNC. Thetetra-tricopeptide repeat (TPR) domain of TgUNC was not essential toobtain fully functional myosin. It has now been identified that purifiedTgMyoA heavy chain complexed with its regulatory light chain (TgMLC1)moved actin in an ensemble motility assay at a speed of ˜1.5 μm/s. Whena putative essential light chain (TgELC1) was also co-expressed, TgMyoAmoved actin at more than twice that speed (˜3.4 μm/s). This indicatedthat both light chains bind to and stabilize the lever arm, which is themyosin domain that amplifies small motions at the active site into thelarger motions needed to propel actin at fast speeds. These methodsresulted in successful expression of milligram quantities of a class XIVmyosin in a heterologous system, and the methods and the prepared classXIV myosin can be used to perform both detailed structure-functionanalysis of TgMyoA and to identify compounds that inhibit the class XIVmyosin.

In one aspect the invention includes methods for producing a functionalclass XIV myosin polypeptide. The method in some embodiments may includeco-expressing three or more polynucleotides in an expression-systemcell, wherein the three or more polynucleotides comprise a class XIVheavy chain polypeptide-encoding polynucleotide, one or more myosinlight chain polypeptide-encoding polynucleotides, and a parasiteco-chaperone polypeptide-encoding polynucleotide. In certain embodimentsof the invention, the three or more polynucleotides are co-expressed inthe cell under conditions suitable to produce a functional class XIVmyosin polypeptide comprising the class XIV heavy chain polypeptide andthe first myosin light chain polypeptide.

Myosin Components

Some aspects of the invention include methods of preparing a functionalclass XIV myosin polypeptide. The term “class XIV myosin” as usedherein, means a class XIV myosin heavy chain polypeptide complexed withat least one of a regulatory light chain (MLC1) and an essential lightchain (ELC1). As used herein the term “complexed” or “complex” used inconjunction with myosin means binding together of various componentssuch as a heavy chain and one or more light chain polypeptides to makeup a class XIV myosin. For example, one or more light chains may bind toa myosin heavy chain, thus forming a myosin complex. Additionalcomponents and/or molecules may be included in a myosin complex include,but are not limited to actin, calmodulin, glideosome polypeptide, etc.

In certain embodiments of the invention, a functional myosin comprises aclass XIV myosin heavy chain polypeptide complexed with an MLC1polypeptide. In certain embodiments, a functional myosin of theinvention comprises a class XIV myosin heavy chain polypeptide complexedwith an ELC1 polypeptide. In certain embodiments, a functional myosin ofthe invention comprises a class XIV myosin heavy chain polypeptidecomplexed with an MLC1 polypeptide and an ELC1 polypeptide. MLC1 andELC1 polypeptides may be collectively referred to herein as “light chainpolypeptides”.

Heavy Chain

Prepared functional myosins of the invention may in some embodimentscomprise a sequence of a Plasmodium falciparum class XIV myosin heavychain or a variant thereof. A non-limiting example of a nucleotidesequence that encodes a Plasmodium falciparum polypeptide referred toherein as a PfMyoA heavy chain, is set forth herein as SEQ ID NO:1,which encodes the Plasmodium falciparum class XIV heavy chainpolypeptide having an amino acid sequence set forth herein as SEQ IDNO:2. In some embodiments of the invention, the heavy chain in aprepared functional class XIV myosin of the invention comprises asequence of a Toxomplasma gondii class XIV myosin heavy chain or avariant thereof. A non-limiting example of a nucleotide sequence thatencodes a Toxomplasma gondii polypeptide referred to herein as a TgMyoAheavy chain, is set forth herein as SEQ ID NO:15, which encodes theToxomplasma gondii class XIV heavy chain polypeptide having an aminoacid sequence set forth herein as SEQ ID NO:16. Sequences fromadditional apicomplexan organisms may also be used in methods of theinvention to prepare class XIV myosins. Non-limiting examples of classXIV heavy chain polypeptide-encoding polynucleotides that may be used inmethods of the invention to prepare a class XIV myosin include, but arenot limited to polynucleotides that encode a Toxoplasma, Plasmodium,Neospora, Sarcocystis, Eimeria, or Cryptosporidium class XIV heavy chainpolypeptide or a functional variant thereof.

Light Chains

Prepared functional myosins of the invention may in some embodimentscomprise one, two, or more sequences of a light chain polypeptide or avariant thereof. As described herein, a light chain may be a“regulatory” light chain, referred to herein as an MLC1 polypeptide,which is also referred to in P. falciparum as Myosin A tail domaininteracting protein (MTIP). In some embodiments of the invention, aregulatory light chain comprises the sequence of a Plasmodium falciparumregulatory light chain or a variant thereof. A non-limiting example of anucleotide sequence that encodes a Plasmodium falciparum regulatorylight chain polypeptide referred to herein as a MTIP light chain, is setforth herein as SEQ ID NO:5, which encodes the Plasmodium falciparumregulatory light chain polypeptide having an amino acid sequence setforth herein as SEQ ID NO:6. In some embodiments of the invention, theregulatory light chain in a prepared functional class XIV myosin of theinvention comprises a sequence of a Toxomplasma gondii regulatory lightchain or a variant thereof. A non-limiting example of a nucleotidesequence that encodes a Toxomplasma gondii polypeptide referred toherein as a MLC1, is set forth herein as SEQ ID NO:17, which encodes aToxomplasma gondii MLC1 light chain polypeptide having an amino acidsequence set forth herein as SEQ ID NO:18.

As described herein, a light chain may be an “essential” light chain,referred to herein as an ELC1 polypeptide. In some embodiments of theinvention, an essential light chain comprises the sequence of aPlasmodium falciparum essential light chain, or a variant thereof. Anon-limiting example of a nucleotide sequence that encodes a Plasmodiumfalciparum essential light chain polypeptide referred to herein as anELC1 light chain, is set forth herein as SEQ ID NO:7, which encodes thePlasmodium falciparum essential light chain polypeptide having an aminoacid sequence set forth herein as SEQ ID NO:8. Another non-limitingexample of a nucleotide sequence that encodes a Plasmodium falciparumessential type light chain polypeptide, which may also be referred toherein as an ELC1 light chain, is set forth herein as SEQ ID NO:50,which encodes the Plasmodium falciparum essential light chainpolypeptide having an amino acid sequence set forth herein as SEQ IDNO:49. In some embodiments of the invention, the essential light chainin a prepared functional class XIV myosin of the invention comprises asequence of a Toxomplasma gondii essential light chain or a variantthereof. A non-limiting example of a nucleotide sequence that encodes aToxomplasma gondii essential light chain polypeptide, is set forthherein as SEQ ID NO:47, which encodes a Toxomplasma gondii essentiallight chain polypeptide having an amino acid sequence set forth hereinas SEQ ID NO:48.

In certain embodiments of the invention, sequences from additionalapicomplexan organisms may be used in methods of the invention toprepare class XIV myosins. Non-limiting examples of regulatory and/oressential light chain polypeptide-encoding polynucleotides that may beused in methods of the invention to prepare a class XIV myosin include,but are not limited to polynucleotides that encode a Toxoplasma,Plasmodium, Neospora, Sarcocystis, Eimeria, or Cryptosporidium lightchain polypeptide or a functional variant thereof.

It will be understood that each apicomplexan polypeptide, or variantthereof, that is included in a class XIV myosin prepared using a methodof the invention may be independently selected relative to otherpolypeptides included in the prepared functional class XIV myosin. Forexample, although not intended to be limiting, in certain embodiments ofthe invention a class XIV myosin heavy chain sequence may be aToxoplasma sequence, or functional variant thereof; a MLC1 sequence maybe a Plasmodium sequence, or functional variant thereof; and an ELC1sequence may be a Neospora sequence or functional variant thereof. Inadditional non-limiting examples, in certain embodiments of theinvention, each of the class XIV myosin heavy chain sequence, a MLC1sequence, and/or an ELC1 sequence may be a Plasmodium sequence, orfunctional variant thereof; each may be a Toxoplasma sequence, orfunctional variant thereof; each may be an Eimeria sequence, orfunctional variant thereof, etc.

Additional Polypeptide Components

In certain aspects of the invention, methods of preparing a functionalclass XIV myosin includes expressing one or more additional polypeptideswith a class XIV myosin heavy chain polypeptide and one or more lightchain polypeptides. Examples of additional types of polypeptides thatmay be expressed with the heavy chain and one or more light chainsinclude, but are not limited to calmodulin polypeptides and GlideosomeAssociated Protein-45 (GAP45) polypeptides. The TgMyoA motor is locatedbetween the plasma membrane and the inner membrane complex (IMC), adouble membrane that is continuous around most of the cell. TgGAP50 (a50 kDa gliding associated protein), an integral membrane glycoprotein ofthe IMC, acts as a membrane receptor for the motor. TgMyoA is linkedindirectly to TgGAP50 through an apicomplexan-specific N-terminalextension of its regulatory light chain, TgMLC1, and TgGAP45 (a 45 kDagliding associated protein). A similar linkage exists in otherapicomplexan parasites, for example with PfMyoA, PfMTIP, PfGAP45, andPfGAP50 in plasmodium falciparum, and equivalent polypeptides present inother apicomplexan parasites MyoA complexes. Thus, certain embodimentsof the invention may include expression of a GAP45 polypeptide. Suchexpression may enable an expressed myosin motor complex to express anincreased level of activity. In certain embodiments of the invention,inclusion of a GAP45 polypeptide may be useful in methods and systems ofthe invention to screen and determine class XIV myosin activity, and inmethods of the invention to screen for and to assess a candidate drug oragent's ability to alter activity of a functional class XIV myosin.Disruption of the connection between the MyoA motor and GAP45, mediatedby TgMLC1 (or PfMTIP) may also be detrimental for glideosome function.

In some embodiments a calmodulin polypeptide comprises an amino acidsequence of a Plasmodium falciparum putative calmodulin sequence such asthe amino acid sequence set forth herein as SEQ ID NO:10, or a variantthereof. The polypeptide set forth as SEQ ID NO:10 may be encoded by anucleotide sequence set forth herein as SEQ ID NO:9. In some embodimentsthe calmodulin polypeptide comprises an amino acid sequence of aToxoplasma gondii putative calmodulin sequence such as the amino acidsequence set forth herein as SEQ ID NO: 20, or a variant thereof. Thepolypeptide set forth as SEQ ID NO:20 may be encoded by a nucleotidesequence set forth herein as SEQ ID NO:19. Calmodulin (and putativecalmodulin) sequences, and variants thereof, from additionalapicomplexan organisms may also be used in methods of the invention toprepare class XIV myosins. Non-limiting examples of calmodulinpolypeptide-encoding polynucleotides that may be expressed with a myosinheavy chain and light chain(s) in methods of the invention to prepare aclass XIV myosin include, but are not limited to polynucleotides thatencode a Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria, orCryptosporidium calmodulin polypeptide or functional variant thereof.

In some embodiments a and glideosome Associated Protein-45 (GAP45)polypeptide comprises an amino acid sequence of a Plasmodium falciparumGAP45 sequence such as the amino acid sequence set forth herein as SEQID NO:44, or a variant thereof. The polypeptide set forth as SEQ IDNO:44 may be encoded by a nucleotide sequence set forth herein as SEQ IDNO:43. In some embodiments the GAP45 polypeptide comprises an amino acidsequence of a Toxomplasma gondii GAP45 sequence such as the amino acidsequence set forth herein as SEQ ID NO:46, or a variant thereof. Thepolypeptide set forth as SEQ ID NO:46 may be encoded by a nucleotidesequence set forth herein as SEQ ID NO:45. GAP45 sequences, and variantsthereof, from additional apicomplexan organisms may also be used inmethods of the invention to prepare class XIV myosins. Non-limitingexamples of GAP45 polypeptide-encoding polynucleotides that may beexpressed with a myosin heavy chain and light chain(s) in methods of theinvention to prepare a class XIV myosin include, but are not limited topolynucleotides that encode a Toxoplasma, Plasmodium, Neospora,Sarcocystis, Eimeria, or Cryptosporidium GAP45 polypeptide or functionalvariant thereof.

Co-Chaperone Molecules

It has now been discovered that by co-expressing a class XIV myosinheavy chain polypeptide, one or more light chain polypeptide(s), and aparasite co-chaperone in a suitable cell system a functional class XIVmyosin can be prepared. As used herein, the term “parasite co-chaperone”means a myosin co-chaperone that is a member of the UCS(UNC-45/Cro1/She4p) family. The UCS family is a group of proteins thatare necessary for a variety of myosin- and actin-dependent functions ineukaryotic organisms. Methods of the invention, in some aspects includeco-expression of a class XIV myosin heavy chain and at least one lightchain with a member of the UCS family or a variant thereof.

In some embodiments of the invention, a UCS family co-chaperone that isexpressed with a class XIV myosin heavy chain and one or more lightchains is a UNC polypeptide variant. In some embodiments of theinvention, a chaperone polypeptide encoding polynucleotide sequencecomprises a homolog of the sequence of the C. elegans UNC-45 sequenceand may be derived from a Toxoplasma, Plasmodium, Neospora, Sarcocystis,Eimeria, or Cryptosporidium organism.

In some embodiments the UNC polypeptide comprises an amino acid sequenceof a Plasmodium falciparum UNC sequence such as the amino acid sequenceset forth herein as SEQ ID NO:4, or a variant thereof. The polypeptideset forth as SEQ ID NO:4 may be encoded by a nucleotide sequence setforth herein as SEQ ID NO:3. In some embodiments the UNC polypeptidecomprises an amino acid sequence of a Toxomplasma gondii UNC sequencesuch as the amino acid sequence set forth herein as SEQ ID NO: 12, or avariant thereof. The polypeptide set forth as SEQ ID NO:12 may beencoded by a nucleotide sequence set forth herein as SEQ ID NO:11. Incertain embodiments of the invention, a truncated UNC polypeptide may beused to prepare a class XIV myosin using methods of the invention. Anon-limiting example of a truncated UNC polypeptide, and its encodingnucleotide are set forth herein as SEQ ID NO:14 and SEQ ID NO:13,respectively. UNC sequences, and variants thereof, from additionalapicomplexan organisms may also be used in methods of the invention toprepare class XIV myosins. Non-limiting examples of UNCpolypeptide-encoding polynucleotides that may be used as a co-chaperonein methods of the invention to prepare a class XIV myosin include, butare not limited to polynucleotides that encode a Toxoplasma, Plasmodium,Neospora, Sarcocystis, Eimeria, or Cryptosporidium UNC polypeptide orfunctional variant thereof.

Labels/Tags

Expression constructs used in methods of the invention may comprise oneor more of a detectable label, also referred to herein as a “tag”. Thus,in certain embodiments, one or more polynucleotides that each encodes apolypeptide label may be included in a construct of the invention.Examples of polypeptide labels that may be encoded by a polynucleotidesequence included in a construct of the invention include, but are notlimited to: a FLAG tag, a biotin acceptor site, a Myc tag, a His tag, ora Ty1 tag. Using standard methods, a skilled artisan will be able toidentify and utilize additional types of detectable labels inembodiments of methods and constructs of the invention.

Expressed Polypeptides

It will be understood that various combinations of the polypeptidecomponents described herein as being possible to include in a preparedmyosin of the invention may be utilized in methods of the invention andin myosins prepared using such methods. In a non-limiting example,expression of a class XIV myosin polypeptide with a parasiteco-chaperone may be suitable to prepare a class XIV myosin for assayssuch as structural assessment using crystallography, etc. Similarly, inother non-limiting examples methods of the invention, in someembodiments may include (1) expression of a class XIV myosinpolypeptide, a parasite co-chaperone polypeptide and a regulatory myosinlight chain polypeptide; (2) expression of a class XIV myosinpolypeptide, a parasite co-chaperone polypeptide, and an essentialmyosin light chain polypeptide; (3) expression of a class XIV myosinpolypeptide, a parasite co-chaperone polypeptide, a regulatory myosinlight chain polypeptide, and an essential myosin light chainpolypeptide; (4) expression of a class XIV myosin polypeptide, aparasite co-chaperone polypeptide, a regulatory myosin light chainpolypeptide, and calmodulin; (5) expression of a class XIV myosinpolypeptide, a parasite co-chaperone polypeptide; an essential myosinlight chain polypeptide, and calmodulin; etc. Additional combinations ofthe components including, but not limited to those described herein maybe used in embodiments of methods of the invention.

Polynucleotides, Polypeptides, and Modified/Variant Sequences

One aspect of the invention involves use of nucleic acid molecules thatencode components of a functional class XIV myosin, a co-chaperone, oranother polypeptide that may be complexed with a class XIV myosin asprepared using methods of the invention. As used herein, the terms“nucleic acid molecule” or “polynucleotide” are intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA). Anucleic acid molecule may be single-stranded or double-stranded or maybe a double-stranded DNA molecule. An “isolated” nucleic acid moleculeis free of sequences that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,may be free of other cellular material.

The invention further encompasses nucleic acid molecules that differfrom the sequences set forth herein for a class XIV myosin heavy chain,light chains, UNC, calmodulin, GAP45, etc. (and portions thereof) due todegeneracy of the genetic code and thus encode the same polypeptide asthat encoded by the disclosed sequences. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequence asset forth herein as a class XIV myosin heavy chain, light chains, UNC,calmodulin, GAP45, etc. (and portions thereof).

Aspects of the invention may include expression of class XIV myosinpolypeptide sequences. Non-limiting examples of polypeptide sequencesthat may be used in embodiments of methods of the invention, include aclass XIV myosin heavy chain polypeptide, a light chain, a UNCpolypeptide, calmodulin, GAP45, etc. Such polypeptide sequences maycomprise a wild-type polypeptide sequence or may be a modifiedpolypeptide sequence that is a variant of a wild-type sequence.

As used herein the term “modified” or “modification” in reference to avariant nucleic acid or polypeptide sequence refers to a change of one,two, three, four, five, six, or more nucleotides or amino acids in thesequence as compared to the sequence from which it was derived. In anon-limiting example, a modified polypeptide sequence may be identicalto a wild-type polypeptide sequence except that it has one, two, three,four, five, or more amino acid substitutions, deletions, insertions, orcombinations thereof. In some embodiments a modified or variant sequencemay be a truncated sequence that is shorter than the sequence from whichit was derived. As used herein the term “derived” and “derived from” maymean a specific sequence may be obtained from a particular source albeitnot necessarily directly from that source. In some embodiments of theinvention a modified or variant sequence may include one, two, three,four, or more amino acid substitutions in a wild-type apicomplexansequence. The term “functional variant” as used herein means a modifiedor variant sequence that retains at least a portion of the function oractivity of the sequence from which it was derived. It will beunderstood that sequences of functional class XIV myosins as preparedusing methods of the invention may be derived from various members ofthe apicomplexan family and may be independently selected relative tothe other sequences used in the methods. Non-limiting examples ofdifferent wild-type amino acid sequences and nucleotide sequences andvariant sequences are provided herein.

Routine sequence alignment methods and techniques can be used to aligntwo or more substantially similar apicomplexan sequences, including butnot limited to sequences from Toxoplasma, Plasmodium, Neospora,Sarcocystis, Eimeria, or Cryptosporidium, etc., thus providing a meansby which a corresponding location of a modification made in one sequencecan be identified in another corresponding apicomplexan sequence.

A polypeptide sequence used in methods of the invention, for example aheavy chain, a light chain, an UNC polypeptide, calmodulin, GAP45, etc.may include amino acid variants (e.g., polypeptides having a modifiedsequence) of the naturally occurring wild-type sequences, as set forthherein. Modified polypeptide sequences may have at least about 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% residue similarity to thepolypeptide sequence disclosed herein. Methods of the invention may insome embodiments, include use of homologs of polypeptides disclosedherein. Such sequence homology can be determined using standardtechniques known in the art. Polypeptide and nucleotide sequences usefulin embodiments of methods of the present invention include thepolypeptide and nucleotide sequences provided herein and variants thathave more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, or 98% sequence similarity to a provided sequence. The percentsimilarity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % similarity=numberof identical positions/total number of positions×100). Such an alignmentcan be performed using any one of a number of well-known computeralgorithms designed and used in the art for such a purpose.

Polypeptides useful in embodiments of the invention may be shorter orlonger than their corresponding polypeptide sequences set forth herein.Thus, in some embodiments, a polypeptide included in a functional classXIV myosin is a full-length polypeptide or a functional fragmentthereof. Similarly, one or more of a class XIV myosin heavy chain, alight chain, an UNC polypeptide, calmodulin, GAP45, or otherpolypeptides used in methods of the invention may be full-lengthpolypeptides or functional fragments thereof. A fragment of apolypeptide may also be referred to herein as a truncated polypeptide.

In some aspects of the invention, substantially similar apicomplexanpolypeptide sequences may have at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or 100% similarity to an apicomplexansequence disclosed herein. Art-known alignment methods and tools can beused to align substantially similar sequences permitting positionalidentification of amino acids that may be modified as described hereinto prepare additional polypeptides useful in methods of the invention toprepare functional class XIV myosin.

Sequence modifications can be in one or more of three classes:substitutions, insertions or deletions. These modified sequences, (whichmay also be referred to as variants) ordinarily are prepared by sitespecific mutagenesis of nucleic acids in the DNA encoding a polypeptide,using cassette or PCR mutagenesis or other techniques known in the art,to produce DNA encoding the modified polypeptide, and thereafterexpressing the DNA in recombinant cell culture. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the polypeptides used in various embodimentsof the invention. Modified polypeptides use in embodiments of theinvention generally exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics.

A site or region for introducing an amino acid sequence modification maybe predetermined, and the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed modified polypeptide screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample, M13 primer mutagenesis and PCR mutagenesis.

Amino acid substitutions are typically of single residues; andinsertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions may range from about 1 to about 20 residues, although in somecases deletions may be much larger. Substitutions, deletions, insertionsor any combination thereof may be used to arrive at a final variantpolypeptide used in methods and functional class XIV myosin of theinvention. Generally these changes are done on a few amino acids tominimize the alteration of the molecule. However, larger changes may betolerated in certain circumstances.

In some embodiments of the invention conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a polypeptide may be replaced with another aminoacid residue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of apolypeptide coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for activity of the resultingpolypeptide to identify mutants that retain activity when expressed toprepare a class XIV myosin in an embodiment of a method of theinvention. Following mutagenesis of a sequence such as a nucleotide orpolypeptide sequence set forth herein, the encoded protein can beexpressed with components to prepare a class XIV myosin as describedherein and the activity of the polypeptide can be determined, forexample using an assay such as those described herein to assess functionand activity of a prepared class XIV myosin.

Variants of polypeptides set forth herein, may exhibit the samequalitative class XIV myosin functionality activity as one or more ofthe sequences set forth herein, such as a class XIV myosin heavy chainpolypeptide, a regulatory light chain polypeptide, an essential lightchain polypeptide, a UNC polypeptide, a calmodulin polypeptide, etc. butmay show some altered characteristics such as altered speed of actinmovement, recovery, compatibility, and toxicity, or a combinationthereof. For example, the polypeptide can be modified such that whenused to prepare a class XIV myosin, the prepared class XIV myosin has anincreased speed of actin movement or a higher level of complexing thanwhen using another polypeptide.

A polypeptide of the invention such as a class XIV myosin heavy chain, aregulatory light chain, an essential light chain, a UNC polypeptide, acalmodulin polypeptide, etc. can incorporate unnatural amino acids aswell as natural amino acids. An unnatural amino acid can be included ina polypeptide of the invention to enhance a characteristic (such ofspeed of actin movement, etc.) of a class XIV myosin that prepared withthe polypeptide. Some embodiments of the invention include compositionsthat include one or more polypeptides of the invention, including butnot limited to: a class XIV myosin heavy chain, a regulatory lightchain, an essential light chain, a UNC polypeptide, and a calmodulinpolypeptide. A composition may also include a carrier such as a buffer,solvent, solute, etc.

Another aspect of the invention provides nucleic acid sequences thatcode for a polypeptide used in methods of the invention to prepare aclass XIV myosin. It would be understood by a person of skill in the artthat the polypeptides of the present invention can be coded for byvarious nucleic acids. Each amino acid in the polypeptide is representedby one or more sets of 3 nucleic acids (codons). Because many aminoacids are represented by more than one codon, there is not a uniquenucleic acid sequence that codes for a given protein. It is wellunderstood by those of skill in the art how to make a nucleic acid thatcan code for a polypeptide of the invention by knowing the amino acidsequence of the protein. A nucleic acid sequence that codes for apolypeptide or protein is the “gene” of that polypeptide or protein. Agene can be RNA, DNA, or other nucleic acid than will code for thepolypeptide. Some embodiments of the invention include compositions thatinclude one or more nucleic acid sequences that encode a polypeptide ofthe invention, including but not limited to: a class XIV myosin heavychain, a regulatory light chain, an essential light chain, a UNCpolypeptide, and a calmodulin polypeptide. A composition may alsoinclude a carrier such as a buffer, solvent, solute, etc.

Amino acid and/or polynucleotide sequences that may be used in methodsand expression systems of the invention, include, but are not limited tosequences that are derived from Toxoplasma, Plasmodium, Neospora,Sarcocystis, Eimeria, or Cryptosporidium amino acid and/or nucleic acidsequences. Sequences derived from sequences of various types ofapicomplexan organisms can be used in methods and systems of theinvention, including but not limited to Toxoplasma, including but notlimited to Toxoplasma gondii; Plasmodium, including but not limited toPlasmodium falciparum, P. vivax, P. knowlesi. P. ovale, or P. malariae;Neospora, including but not limited to Neospora caninu or Neosporahughesi; Sarcocystis, including but not limited to Sarcocystisl neurona,bovihominis (S. hominis,), or S. suihominis; Eimeria, including but notlimited to Eimeria tenella, E. bovis, E. necatrix, E. ellipsoidalis, orE. zuernii; and Cryptosporidium, including but not limited toCryptosporidium parvum, C. hominis, C. canis, C. felis, C. meleagridis,or C. muris. Those skilled in the art will readily recognize how toutilize sequences from these types of organisms to prepare functionalclass XIV myosin polypeptide using methods and systems of the invention.The term “protein” and “polypeptide” are used interchangeably herein.

Expression Systems Vectors

Methods of the invention, in some embodiments, include use ofrecombinant expression systems. In embodiments of the invention,expression systems comprises host cells (prokaryotic or eukaryoticcells) that are transformed by vectors and recombinants and that arecapable of expressing said RNA and/or DNA fragments. In certain aspects,the invention provides methods for preparing a functional class XIVmyosin polypeptide comprising the steps of: (a) culturing a host cellcontaining a vector under conditions that provide for expression of thefunctional class XIV myosin polypeptide and (b) recovering the expressedfunctional class XIV myosin polypeptide sequence. This method, in someembodiments, may also include additional methods of (c) subjecting theprepared polypeptide to protein purification.

Some aspects of the invention also relate to methods forproduction/preparation of a recombinant functional class XIV myosinpolypeptide, wherein the methods may include steps of: a) transformingan appropriate cellular host with one or more recombinant vectors, inwhich at least a class IX myosin heavy chain polynucleotide sequence orfunctional fragment thereof, a co-chaperone polynucleotide sequence orfunctional fragment thereof, and one or more light chain polynucleotidesequences or functional fragments thereof, have been inserted under thecontrol of appropriate regulatory elements, b) culturing the transformedcellular host under conditions suitable for the expression of theinsert, and, c) harvesting (e.g., isolating) a class XIV myosinpolypeptide.

Vectors provided by the present invention may typically comprise a classIX myosin heavy chain polynucleotide sequence or functional fragmentthereof, a co-chaperone polynucleotide sequence or functional fragmentthereof, and one or more light chain polynucleotide sequences orfunctional fragments thereof encoding the desired amino acid sequenceand preferably transcription and translational regulatory sequencesoperably linked to the amino acid encoding sequences so as to allow forthe expression of the class XIV myosin polypeptide in the cell. In someembodiments of the invention, a vector will include appropriateprokaryotic, eukaryotic or viral promoter sequence followed by anucleotide sequence as defined above. The recombinant vector of theinvention may allow the expression of a class XIV myosin polypeptide ina prokaryotic, or eukaryotic host or in living mammals when injected asnaked RNA or DNA.

The vector may comprise a plasmid, a cosmid, a phage, or a virus or atransgenic animal. In some embodiments of the invention, the vector is abaculovirus vector. Examples of such expression vectors and insect cellexpression systems and methods are described in The BaculovirusExpression System: A Laboratory Guide, Linda King, Springer; 2012, thecontent of which is incorporated by reference herein. Many usefulvectors are known in the art and may be obtained from such vendors asStratagene, New England Biolabs, Promega Biotech, and others.

In certain embodiments of the invention vectors may include regulatorycontrol sequences that allow for inducible expression of a class XIVmyosin polypeptide, for example in response to the administration of anexogenous molecule. Alternatively, temporal control of expression of theclass XIV myosin polypeptide may occur by only introducing thepolynucleotide into the cell when it is desired to express thepolypeptide.

Expression vectors may also include, for example, an origin ofreplication or autonomously replicating sequence and expression controlsequences, a promoter, an enhancer and necessary processing informationsites, such as ribosome-binding sites, RNA splice sites, polyadenylationsites, transcriptional terminator sequences, and mRNA stabilizingsequences. Secretion signals may also be included where appropriate.Vectors of the invention may be prepared using methods described hereinand with additional art-known standard recombinant techniques well knownin the art and discussed, for example, in Molecular Cloning: alaboratory manual, Green, M. R., J. Sambrook, 2012 Cold Spring HarborLaboratory Press, 4^(th) Edition; The Baculovirus Expression System: ALaboratory Guide, Linda King, Springer; 2012, the content of which areincorporated by reference herein.

An appropriate promoter and other necessary vector sequences may beselected to be functional in the host, and may include, whenappropriate, those naturally associated with apicomplexan (or otherorganism) sequences.

Expression and cloning vectors useful in embodiments of methods of theinvention may contain a selectable marker, a gene encoding a proteinnecessary for survival or growth of a host cell transformed with thevector. The presence of this gene ensures growth of only those hostcells that express the inserts. Typical selection genes encode proteinsthat a) confer resistance to antibiotics or other toxic substances, e.g.ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophicdeficiencies, or c) supply critical nutrients not available from complexmedia, e.g., the gene encoding D-alanine racemase for Bacilli. Thechoice of the proper selectable marker will depend on the host cell, andappropriate markers for different hosts are well known in the art.

Vectors containing a class XIV myosin heavy chain polynucleotidesequence or functional fragment thereof, a co-chaperone polynucleotidesequence or functional fragment thereof, and one or more light chainpolynucleotide sequences or functional fragments thereof sequences canbe transcribed in vitro and the resulting RNA introduced into the hostcell by well-known methods, e.g., by injection, or the vectors can beintroduced directly into host cells by methods well known in the art,which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome); and other methods.The introduction of a class XIV myosin heavy chain polynucleotidesequence or functional fragment thereof, a co-chaperone polynucleotidesequence or functional fragment thereof, and one or more light chainpolynucleotide sequences or functional fragments thereof sequences intoa host cell may be achieved by any method known in the art, including,inter alia, those described herein.

In certain embodiments of the invention a class XIV myosin heavy chainpolynucleotide sequence or functional fragment thereof, a co-chaperonepolynucleotide sequence or functional fragment thereof, and one or morelight chain polynucleotide sequences or functional fragments thereof arepart of a viral vector, such as a baculovirus vector, or infectiousvirus, such as a baculovirus. In certain embodiments of the invention,one, two, three, or more vectors are inserted into a cell for expressionand in some embodiments of the invention, a viral transfer vector may beused that allows 1, 2, 3, 4, 5, or more polynucleotide sequences that apolypeptide to be inserted into the same vector so they can beco-expressed by the same recombinant virus.

Host Cells

To produce a cell capable of expressing a functional class XIV myosinpolypeptide, polynucleotide sequences such as those that encode a classXIV myosin heavy chain polypeptide, a co-chaperone polypeptide, acalmodulin polypeptide, a GAP45 polypeptide, or one or more light chainpolypeptides are incorporated into a recombinant vector, which is thenintroduced into a host prokaryotic or eukaryotic cell.

The invention, in some aspects, also provides host cells transformed ortransfected with a one or more of an exogenous class XIV myosin heavychain polynucleotide sequence, a co-chaperone polynucleotide sequence, acalmodulin polynucleotide sequence, a GAP45 polynucleotide sequence, orone or more light chain polynucleotide sequences. A host cell useful inembodiments of the invention may include but are not limited to a cellfrom yeast, filamentous fungi, a plant, insect, amphibian, avianspecies, bacteria, mammals, and human cells in tissue culture. Incertain embodiments of the invention, a host cell may be a cell from E.coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, COS 1, COS7, BSC1, BSC40, or BMT10 cells. In certain embodiments of the invention,a host cell is an Sf9 cell.

Large quantities of class XIV myosin polypeptide produced/prepared byexpressing a class XIV myosin heavy chain polynucleotide sequence with aco-chaperone polynucleotide sequence and optionally with one or more ofa calmodulin polynucleotide sequence, a GAP45 polynucleotide sequence,one or more light chain polynucleotide sequences—(or fragments of any ofthe listed polynucleotide sequences) in vectors or other expressionvehicles in compatible prokaryotic or eukaryotic host cells.

In certain embodiments of the invention a prepared class XIV myosinpolypeptide is isolated. As used herein, the term “isolated” when usedin reference to a prepared polypeptide of the invention includes suchpolypeptides that are separated from the system in which they areexpressed. Isolated prepared class XIV myosin polypeptides may be usedin various assays to determine and measure activity of the myosin, totest the effect of contacting the myosin polypeptide with one or morecandidate compounds, to assess function of the myosin polypeptide, etc.

Function and Assays

As used herein the term “functional” when used in regard to a class XIVmyosin polypeptide means a class XIV myosin polypeptide that undersuitable conditions will move actin. Actin movement may be detected andassessed using assays, such as, but not limited to, an ensemble motilityassay. Such assays can be used to assess whether a prepared myosinpolypeptide is functional, and also to determine a speed at which thefunctional myosin moves actin. In some embodiments a functional classXIV myosin moves actin at a speed of up to about 0.5, 1.0, 1.5, 2.0,2.5, 3.0, 3.5, or 4.0 μm/s.

A number of different assays may be used to assess function of preparedclass XIV myosin. Non-limiting examples include actin-activated APTaseassays, which may include sensitive colorimetric methods that usemalachite green and are compatible with high throughput plate screening,for example, 96-well plate screening. See for example Henkel, R. D., etal., (1988) Anal. Biochem. 169, 312-318. Actin pelleting methods mayalso be used in some aspects may include centrifugation of actin andmyosin for 25 min at 350,000×g or other suitable conditions. Followingcentrifugation, myosin bound to actin is quantified using a method suchas SDS-gel densitometry or by protein concentration determination in thesupernatant. Another non-limiting example of an activity assay is an invitro motility assay, as described in Trybus, K. M. (2000) Methods 22,327-335. Additional assays are also suitable for determining andassessing function of a class XIV myosin, such as one prepared using amethod of the invention, or other control class XIV myosins.

Methods of Activity/function Assessment

It has been identified that class XIV myosin activity may contribute toonset of infection of a subject by a parasite such as an apicomplexanorganism. Function class XIV myosin prepared using methods of theinvention may be useful in assays to identify compounds useful toprevent or treat an infection of a subject by a parasite such as anapicomplexan parasite. A compound that when contacted with a functionalclass XIV myosin reduces the myosin's activity by at least 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, may be a compounduseful to prevent or treat an apicomplexan infection or to preventcontamination of a substrate by an apicomplexan organism. Methods of theinvention, in some embodiments, include methods of identifying acompound as a candidate compound to inhibit a parasite that expresses aclass XIC myosin polypeptide.

In certain embodiments of the invention, prepared myosin of theinvention may be used to assess compounds that when contacted with afunctional class XIV myosin increase the myosin's activity by at least1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or moreand thus enhance one or more functions or activities of an apicomplexanorganism that expresses a class XIV myosin. Thus, the invention, in someembodiments, may include methods of identifying a compound as acandidate compound to enhance a parasite that expresses a class XIVmyosin polypeptide.

Identification of Candidate Compounds

The invention, in some aspects also includes methods to identifycandidate compounds that decrease (or increase) class XIV myosinactivity when contacted with a cell, tissue, subject, or substrate. Theinvention, in part, may also include methods to assess the efficacy ofcandidate class XIV myosin activity-modulating compounds to decrease thelevel of expression and/or to reduce the level of an activity/functionof a class XIV myosin polypeptide in a cell or organism. Such methodsmay be carried out in vivo in human or animal subjects; or using invitro assays of the invention such as in cells from culture—e.g., asscreening assays to assess compounds that may—modulate class XIV myosindirectly or indirectly, for example by modifying level or activity of aco-chaperone, or other molecule. Such modulating compounds that directlyor indirectly alter class XIV myosin polypeptide activity in a cell,tissue, or organism may be used in the treatment or prevention of anapicomplexan infection or contamination of a substrate by anapicomplexan organism.

Assays for assessing activity levels of a prepared class XIV myosin inembodiments of the invention may include determining one or more classXIV myosin levels and/or activities, including but not limited todetermining levels of function/activity of components of a class XIVmyosin polypeptide (e.g., a heavy chain, a light chain), or otherpolypeptides described herein such as a co-chaperone, calmodulin, aGAP45 polypeptide etc. that are co-expressed in methods of the inventionuseful to prepare a functional class XIV myosin. For example, assaymethods of the invention may be used to determine whether a candidatecompound interferes with (e.g., reduces) an interaction between a myosinheavy chain polypeptide and one or more light chains. In anothernon-limiting example of assays for candidate compounds, methods of theinvention may also be used to determine whether a candidate compoundinterferes (e.g., reduces) an interaction between a co-chaperone andfolding of the heavy chain. Such interference may reduce thefunction/activity of the prepared class XIV myosin and thus, contactingan apicomplexan organism with the compound may be useful to reduce theorganism's ability to cause an infection and this prevent or treat anapicomplexan infection of a subject or apicomplexan contamination of asubstrate.

As used herein, aspects of the invention may include assessment ofinteractions between component polypeptides that are part of a class XIVmyosin complex or are used to prepare a functional class XIV myosin.Examples of such component polypeptides include, but are not limited to,a class XIV myosin heavy chain, a regulatory light chain, an essentiallight chain, calmodulin, a co-chaperone, a UCS polypeptide, a GAP45polypeptide, etc. Assay methods of the invention may be used to assesswhether a candidate compound interferes with interactions between one ormore component polypeptides.

In some embodiments of the invention, a level of activity or interactionof one or more component polypeptides is measured in relation to acontrol level of the activity or interaction of the one or morecomponent polypeptides. One possible measurement of the level acomponent polypeptide may be a measurement of an absolute level of acomponent polypeptide or its encoding nucleotide. This could beexpressed, for example, in the level of a component polypeptide orencoding nucleotide per unit of cells or unit volume in an assay.Another measurement of a level of a component polypeptide or itsencoding nucleotide may be a measurement of the change in the level ofthe component polypeptide or its encoding nucleotide over time. This maybe expressed in an absolute amount or may be expressed in terms of apercentage increase or decrease over time.

In some aspects, the invention includes methods that provide informationon the efficacy of a candidate compound to treat an apicomplexaninfection or contamination. In certain aspects, the invention includesmethods to assess activity and efficacy of compounds administered to asubject to prevent or treat an apicomplexan infection. Similarly, theinvention in some embodiments may include methods to assess activity andefficacy of compounds contacted with an apicomplexan organism.Information about the stage or status of an infection or contaminationby an apicomplexan organism and the efficacy of a compound or treatmentof an infection by the organism can be used to assist a health-careprovided to select a treatment for administration to a subject at riskor known to have an apicomplexan infection or can be used by ahealth-care professional to adjust (e.g., increase, decrease, or stop) atreatment that is being provided to such a subject.

As used herein a “subject” refers to any animal, such as, but notlimited to a human, a non-human primate, a rodent, a dog, cat, bird,horse, or other animal. Thus, in addition to human medical application,some aspects of the invention include veterinary application of methodsdescribed herein.

In some aspects of the invention, methods are provided to identifycandidate compounds for preventing or treating an apicomplexan infectionof a subject and/or to reduce apicomplexan contamination of a substrate.Methods of the invention may be used to determine the efficacy of acompound for prevention and/or treatment of the infection orcontamination. Such methods may include, for example, determining one ormore levels of activity of a prepared functional class XIV myosin andcontacting the class XIV myosin with a candidate compound anddetermining whether there is an increase, decrease, or no change in theactivity of the class XIV myosin.

As described herein, a level of function/activity in a class XIV myosinprepared using methods of the invention can be determined using assaymethods of the invention to measure the amount and/or activity of aprepared class XIV myosin molecule in an in vitro assay. As used herein,the term “measure” may refer to a determination of the presence orabsence of activity of a prepared class XIV myosin and may refer to adetermination of a level of activity of a prepared class XIV myosin.Methods of measuring activity of a class XIV myosin polypeptide areknown in the art, and non-limiting examples of measuring means areprovided herein.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES Example 1 Expression Constructs

Full length (FL) TgUNC was amplified from T. gondii cosmid #p559;(http://toxodb.org/toxo/; cosmid generously provided by Dr. M J Gubbels)using the primer pair TPRForANcoI and TPRRevAFullMycStpKpnI (see Table 1for primer sequences). A Myctagged TgUNC PCR product was cloned into thebaculovirus transfer vector pAcSG2 (BD Biosciences). TgUNC pAcSG2 wastruncated to make two other constructs. TgUNCΔTPR starts at residueLeu161, eliminating the tetra-trico-peptide repeat region (TPR) butkeeping the central and UCS domains along with the C-terminal Myc tag.The TgUNC UCS only construct starts with residue Glu682, thuseliminating both the TPR and central domains.

TABLE 1 List of oligonucleotides used for cloning. Primer SEQ ID Genename Primer sequence NO TgUNC TPR cggcggtaccttacaggtcttc 32 RevAFullMycttcagagatcagtttctgtt STPKpnI cgcttgagtctgg. TPRForANcolagca ccatgg aggatttg 33 tcaaacgc. TgMyoA EcoR1-FLAG-gggggaattcatggactacaa 34 MyoF1 agacgatgacgacaagatggc. c-TgMyoA-gggggaattcctactactag 35 EcoRI aacgccggctgaacagtc. TgMLC1 MLC-Fcatggaattcatgagcaa 36 ggtcgagaaga. MLC-R catgagatcttgattactc 37ccttcgctcgag. 6HISMLC gcacaatcatatgcatca 38 1NdeIF ccaccatcatcacagcaaggtcgagaagaaatgc. MLC1BamHIR gcacaatggatccttactccctt 39 cgctcgagcatt.TgELC1 6HISELC gcacaatcatatgcatcacc 40 1NdeIF accatcatcacacctgccctccccgcgt ccgt. ELC1BamHIR gcacaatggatccttatttcag 41cagcatcttgacaaagt.

The plasmid pEB2-FLAG-MyoA (Lucas Tilley and GEW, unpublished) was usedas a template to amplify nFLAG tagged TgMyoA heavy chain (ToxoDBTgGT1_(—)235470) using primer pair EcoR1-FLAG-MyoF1 and c-TgMyoA-EcoR1.The PCR product was cloned into the baculovirus transfer vector pAcUW51(BD Biosciences). TgMyoA-cBio-cFLAG was constructed by digesting TgMyoAheavy chain from pVL1392FLAGTgMyoA (Aoife Heaslip and GEW, unpublished)with BamHI/EcoRV and inserting the cleavage fragment into a modifiedversion of pAcSG2 containing both a biotin acceptor site and FLAG tag atthe C-terminus.

The TgMLC1 gene (ToxoDB TgGT1_(—)257680) was PCR amplified from parasitecDNA using primers MLC-F and MLC-R and cloned untagged by ligation ofthe digested, gel extracted PCR product at the EcoRI and BglIIrestriction sites in pAcSG2. The 6×His tagged MLC1 gene was amplifiedfrom pAcSG2 TgMLC1 (Aoife Heaslip and GEW, unpublished) using the primerpair 6HISMLCINdeIF and MLC1BamHIR. The resulting PCR product was clonedinto the bacterial expression vector pET3a (Novagen).

The TgELC1 gene (ToxoDB v 7.3 TgGT1_(—)107770) was PCR amplified from T.gondii RH strain cDNA. This entry codes for a 134 aa (˜15 kDa) proteinas described in (6) and can be viewed athttp://v7-3.toxodb.org/toxo.b15/showRecord.do?name=GeneRecordClasses.GeneRecordClass&project_id=ToxoDB&source_id=TGGT1_(—)107770(last accessed Apr. 2, 2014). The PCR product was ligated downstream ofthe p10 promoter in vector pAcUW51 for baculovirus expression. Inaddition, a PCR product of TgELC1 with a 6×His tag using the primer pair6HisELC1NdeIF and ELC1BamHIR was cloned into the bacterial expressionvector pET3a (Novagen).

A dual light chain plasmid was constructed for immunoprecipitationstudies. TgELC1 with a C terminus 3×HA tag was cloned into vectorpAcUW51 downstream of the p10 promoter, while TgMLC1 with an N-terminal3×Myc tag was cloned downstream of the polH promoter. All constructswere sequenced prior to transfection

Co-Immunoprecipitations

Sf9 cells were harvested 72 h after infection with recombinantbaculoviruses (TgMyoA heavy chain and tagged light chains), and lysedwith 10 mM imidazole Ph 7.4, 150 mM NaCl, 1 mM EGTA, 5 mM MgCl2, 7%(w/v) sucrose, 3 mM NaN3, 1% (v/v) NP-40, 1 mM DTT and 1× proteaseinhibitor cocktail (Sigma-Aldrich, catalog# P8340). Following additionof 5 mM MgATP, the lysate was spun at 350,000×g for 20 min at 4° C. Thesupernatant was incubated with either rabbit anti-TgMLC1 (a generousgift from Dr. Con Beckers) or rat anti-HA (Roche #11867423001) overnightat 4° C. Rec-Protein ASepharose beads (Invitrogen) were added and thesamples incubated at 4° C. for 60 min. The beads were washed four timeswith lysis buffer. Bound proteins were eluted by boiling in SDS-PAGEsample buffer, centrifuged at 100×g for 2 min and resolved on 4-12%gradient gels. The protein was transferred to Immobilon-FL (Millipore,Bedford, Mass.) and probed with mouse anti-Myc 9E10 (1:2,000;Developmental Studies Hybridoma Bank, University of Iowa), rat anti-HA(1:400) or rabbit anti-HA (1:2,000, AbCam #9110) and mouse anti-FLAG(1:7,500, Sigma-Aldrich). LI-COR secondary antibodies (anti-MouseIRDye680RD, anti-Rabbit IRDye800CW and anti-Rat IRDye800CW) were usedaccording to manufacturer's instructions and blots were scanned using anOdyssey CLx Infrared Imaging System (LI-COR, Lincoln, Nebr.).

Protein Expression and Purification

Sf9 cells were co-infected with recombinant baculovirus coding forTgMyoA heavy chain (tagged at the Cterminus with a Bio-tag andFLAG-tag), untagged light chain(s), and the co-chaperone TgUNC. Thecells were grown in media supplemented with 0.2 mg/ml biotin. After 72hours, the cells were lysed by sonication in 10 mM imidazole, pH 7.4,0.2 M NaCl, 1 mM EGTA, 5 mM MgCl2, 7% (w/v) sucrose, 2 mM DTT, 0.5 mMAEBSF, 5 μg/ml leupeptin, and 5 mM benzamidine. To determine TgMyoAheavy chain solubility, the extracts were centrifuged at 350,000×g for20 min. For motor purification, 25 μg/ml each of bacterially expressedTgMLC1 and TgELC1 (see below) and 5 mM MgATP were added to the lysate,which was then clarified at 200,000×g for 30 min. The supernatant wasapplied to a FLAG affinity resin column (Sigma-Aldrich) and washed with10 mM imidazole, pH 7.4, 0.2 M NaCl, 1 mM EGTA, and 1 mM NaN3. TgMyoAwas eluted from the column with 0.1 mg/ml FLAG peptide in the columnbuffer. The fractions of interest were combined and concentrated with anAmicon centrifugal filter device (Millipore) and dialyzed against 10 mMimidazole, pH 7.4, 0.2 M NaCl, 50% (v/v) glycerol, 1 mM DTT, and 1 μg/mlleupeptin for storage at −20° C.

HIS-tagged light chains (TgELC1 or TgMLC1) in pET3a (Novagen) wereexpressed in BLR(DE3) competent cells grown in LB broth. The cultureswere induced with 0.4 mM IPTG and grown overnight at 27° C. before beingpelleted and frozen. The pellets were lysed by sonication in 10 mMsodium phosphate, pH 7.4, 0.3 M NaCl, 0.5% (v/v) glycerol, 7% (w/v)sucrose, 7 mM β-mercaptoethanol, 0.5 mM AEBSF, and 5 μg/ml leupeptin.The cell lysate was clarified at 200,000×g for 30 min. TgELC1, which isfound in the supernatant, was boiled for 10 min in a double boiler, andthen clarified at 26,000×g for 30 min. Soluble protein was applied to aHIS-Select® nickel affinity column (Sigma-Aldrich). Non-specificallybound protein was removed by washing the resin with buffer A (10 mMsodium phosphate, pH 7.4, 0.3 M NaCl). TgELC1 was then eluted from thecolumn with buffer A containing 200 mM imidazole. The protein wasdialyzed overnight against 10 mM imidazole, pH 7.4, 150 mM NaCl, 1 mMEGTA, 1 mM MgCl2, and 50% (v/v) glycerol.

Bacterially expressed TgMLC1 is found in the insoluble inclusion bodies.The cell lysate was clarified at 26,000×g for 10 min. The pellet wasdissolved in 20 ml of 8 M guanidine, 150 mM NaCl, 10 mM NaPO₄, pH 7.5,10 mM DTT and stirred at room temperature until dissolved. It was thenclarified at 200,000×g for 30 min and dialysed overnight against 2×1liter of buffer A containing 7 mM β-mercaptoethanol and 1 μg/mlleupeptin. The next day the sample was clarified at 26,000×g for 30 minand the supernatant was applied to a HIS-Select® nickel affinity column(Sigma-Aldrich). The column was washed with 15 ml of dialysis buffer andTgMLC1 was eluted with buffer A containing 200 mM imidazole. The proteinwas dialyzed overnight against 10 mM Imidazole pH 7.4, 150 mM NaCl, 1 mMEGTA, 1 mM MgCl2, and 50% (v/v) glycerol. Both purified light chainswere stored at −20° C.

Gels—

Proteins were separated on a 4-12% Bis-Tris NuPAGE gel (Invitrogen) runin MES buffer, per NuPAGE technical guide.

In Vitro Motility—

To prepare the flow cell, 0.2 mg/ml biotinylated BSA in buffer B (150 mMKCl, 25 mM imidazole, pH 7.5, 1 mM EGTA, 4 mM MgCl₂, 10 mM DTT) wasadded to the nitrocellulose-coated flow cells for 1 min, followed by 3rinses with 0.5 mg/ml BSA in buffer B. Neutravidin (50 μg/ml; ThermoScientific) in buffer B was applied for 1 min, followed by 3 rinses withbuffer B. Before introduction into the flow cell, TgMyoA was mixed witha 2-fold molar excess of F-actin and 10 mM MgATP in buffer B andcentrifuged for 25 min at 350,000×g to remove ATP-insensitive myosinheads. TgMyoA was then introduced into the flow cell at 70 μg/ml. Tofurther block any ATP-insensitive heads, 1 μM vortexed Factin in bufferC (50 mM KCl, 25 mM imidazole, pH 7.5, 1 mM EGTA, 4 mM MgCl₂, 10 mM DTT)was added for 60 s, followed by a 10 mM MgATP wash.Rhodamine-phalloidin-labeled actin was then introduced for 1 min,followed by one rinse with buffer C. Three volumes of Buffer C, whichalso contained 5 mM MgATP (unless stated otherwise), 0.5%-0.7%methylcellulose, 25 μg/ml TgMLC1 and 25 μg/ml TgELC1, 3 mg/ml glucose,0.125 mg/ml glucose oxidase (Sigma-Aldrich), and 0.05 mg/ml catalase(Sigma-Aldrich), were then flowed in. When assayed with calcium, 1.2 mMcalcium was added to this buffer.

Actin movement was observed at 30° C. using an inverted microscope(Zeiss Axiovert 10) equipped with epi-fluorescence, a Rolera MGi PlusDigital camera, and dedicated computer with the Nikon NIS Elementssoftware package. Data were analyzed using a semi-automated filamenttracking program described in (10). The velocities of >600 filamentswere determined. Speeds were fit to a Gaussian curve.

Actin-Activated ATPase Activity—

Assays were performed in 10 mM imidazole, pH 7.0, 5 mM NaCl, 1 mM MgCl₂,1 mM NaN3, and 1 mM DTT at 30° C. Purified TgMyoA (7.5 μg/ml) wasincubated with various concentrations of skeletal actin. Activity wasinitiated by the addition of 5 mM MgATP and stopped with SDS every 2 minfor 8 min. Inorganic phosphate was determined colorimetrically (33). Thelow salt concentration was needed to keep the Km values as low aspossible. Data were fit to the Michaelis-Menten equation.

Sedimentation Velocity—

Sedimentation velocity runs were performed at 20° C. in an Optima XL-Ianalytical ultracentrifuge (Beckman Coulter) using the An60Ti rotor at30,000 rpm. The solvent was 20 mM HEPES, pH 7.4, 0.1 M NaCl, 2 mM DTT.An N-terminally FLAG-tagged TgMyoA heavy chain (no Bio tag) bound toTgMLC1 was used for this experiment. The sedimentation coefficient wasdetermined by curve fitting to one species, using the dc/dt program(34).

Results

TgMyoA Heavy Chain is Insoluble when Expressed in Sf9 Cells—

Recombinant baculoviruses encoding TgMyoA heavy chain and its regulatorylight chain, TgMLC1, were used to co-infect Sf9 cells. The C-terminus ofthe TgMyoA heavy chain contained two tags: a FLAG-tag to facilitatepurification by affinity chromatography, and a Biotag, which becomesbiotinylated within the Sf9 cells (35) and allows the motor to bespecifically attached via its C-terminus to a streptavidin-coatedcoverslip for in vitro motility assays (FIG. 2A). Although both TgMyoAheavy chain and TgMLC1 were expressed following infection, as detectedby Western blotting of the total Sf9 cell lysate, none of the TgMyoAheavy chain was present in the soluble fraction (FIG. 3).

Identification of a UCS Family Gene in the T. gondii Genome—

The studies suggested that the endogenous Sf9 protein folding machinerywas not sufficient to fold this unusual myosin, and that aparasite-specific co-factor was required. A potential candidate was aprotein in the UCS family of myosin co-chaperones, of which UNC-45 fromC. elegans is the founding member. Bioinformatic analysis of the T.gondii genome using either full length Unc45 or the UCS domain revealeda candidate, TgUNC (http://toxodb.org/toxo/; TgGT1_(—)249480). The TgUNCamino acid sequence was aligned with the three canonical members of theUCS family of myosin chaperones (C. elegans Unc45b, P. anserina CRO1,and S. cerevisiae She4p) (FIG. 10). The percent identity/percentsimilarity of full-length TgUNC with the other UCS family members wasdetermined and was found to be: Unc45b, 17.4/28.7; CRO1, 9.5/16.3;She4p, 9.3/17.4. Comparing only the UCS domains, the percentidentity/percent similarity with TgUNC increases to: Unc45b, 20.4/31.1;CRO1, 17.4/27.3; She4p,15.3/25.3. A schematic of the domain structure ofTgUNC is shown in FIG. 2B.

Production of Soluble TgMyoA in Sf9 Cells Requires Co-Expression withTgUNC—

In marked contrast to what was observed in the absence of TgUNC,co-expression of TgMyoA heavy chain and TgMLC1 with TgUNC yieldedsoluble protein in small-scale infections (FIG. 3). A large-scaleinfection for protein purification was set up (500 ml culture, 3×10⁹ Sf9cells) using the same three viruses to co-infect Sf9 cells. TgMyoA waspurified from the lysate using a FLAG-affinity column (see Methods aboveherein). Protein that bound to and eluted from the FLAG column showedtwo predominant bands on SDS-gels at the expected sizes of TgMyoA heavychain and TgMLC1 (FIG. 4A, lane 1). The yield of purified motor was ˜1.5mg/10⁹ Sf9 cells. The isolated expressed protein was analyzed bysedimentation velocity in the analytical ultracentrifuge. The resultsshowed a single symmetrical peak with an S value of 7.75±0.01,indicating a homogeneous preparation of protein (FIG. 4B).

In Vitro Motility of Expressed TgMyoA Containing Bound TgMLC1—

Solubility does not ensure activity, so an in vitro motility assay wasperformed to assess function. TgMyoA with bound gMLC1 was perfused intoa chamber containing a streptavidin-coated coverslip. The biotinylatedtag at the C-terminus of TgMyoA heavy chain ensured that this regionwould adhere to the coverslip, leaving the motor domain accessible tobind actin. Expressed protein moved rhodamine-phalloidin labeledskeletal muscle actin at a speed of 1.5±0.2 μm/s (FIG. 5, solidtriangles). Speeds were calculated using a semi-automated trackingprogram that allowed thousands of trajectories to be analyzed withoutselection bias (10). The speeds fit a Gaussian distribution.

The speed obtained with expressed protein was slower than the speed ofprotein isolated from parasites (8,12), which suggested a need toidentify an additional component of the motor complex.

Co-Expression of TgMyoA Heavy Chain with Both TgMLC1 and TgELC1—

In addition to TgMLC1, an essential light chain called TgELC1 wasrecently identified as part of the myosin motor complex (6). When TgELC1was co-expressed with TgMyoA heavy chain, TgMLC1, and TgUNC, both lightchains were found to co-purify with the heavy chain, indicating that,like TgMLC1, TgELC1 is a tightly bound subunit of TgMyoA. When TgMyoAcontaining both light chains was assayed in the in vitro motility assay,it moved actin at 3.4±0.7 μm/s, more than twice the speed seen withTgMyoA containing only TgMLC1 (FIG. 5, solid circles).

Each TgMyoA Heavy Chain Binds Simultaneously to a Regulatory andEssential Light Chain—

To test whether TgMLC1 and TgELC1 bind simultaneously to the same heavychain, a recombinant baculovirus was prepared that contained both lightchains, each with a different tag. TgMLC1 had an N-terminal 3×Myc tag,while TgELC1 had a C-terminal 3×HA tag. 72 hours following infectionwith heavy and light chains, TgMyoA was immunoprecipitated from the Sf9cell lysate with either an anti-HA antibody (TgELC1) or an anti-TgMLC1antibody (FIG. 6). The Myc antibody could not be used for TgMLC1immunoprecipitation because TgUNC was also tagged with Myc. The eluatesfrom the immunoprecipitation were analyzed by Western blotting withantibodies to detect TgUNC (anti-Myc), TgMyoA heavy chain (anti-FLAG),TgMLC1 (anti-Myc), and TgELC1 (anti-HA). The results showed that theproteins co-immunoprecipitating with TgMLC1 included TgELC1, andconversely that the proteins co-immunoprecipitating with TgELC1 includedTgMLC1. Earlier work showed that individual motor complexes do notphysically associate with each other (12), and it was conclude that bothlight chains are simultaneously present on the TgMyoA heavy chain.

Calcium does not Regulate Motility Speed—

To determine if calcium affects the speed at which TgMyoA moves actin,an in vitro motility assay was performed in the presence or absence offree calcium. TgMyoA was pre-incubated in buffer containing either 0.2mM free calcium or 1 mM EGTA for one hour before performing the assay.Whether the myosin contained TgMLC1 or both light chains, no differencein speed was observed in the presence or absence of calcium (FIG. 5,open symbols).

Steady State Actin-Activated ATPase Activity—

The speed of actin filament movement increased as the MgATPconcentration in the in vitro motility assay increased (FIG. 7A). Thefit to a rectangular hyperbola defined a Vmax of 4.6±0.3 μm/s and a Kmof 1.3±0.3 mM MgATP. Based on this observation, steady-stateactin-activated ATPase assays were performed with 5 mM MgATP. Rates ofATP hydrolysis were determined as a function of skeletal actinconcentration. Data were fit to the Michaelis-Menten equation. TgMyoAexpressed with both light chains showed a Vmax of 84±9.5 s⁻¹ and a Kmfor actin of 136±22 μM at 30° C. (FIG. 7B).

The TPR Domain of TgUNC is not Required to Express Functional Myosin—

Having established the properties of functional TgMyoA, studies wereperformed to determine the minimal domain of TgUNC that produced anactive motor. In addition to full-length TgUNC, two shorter constructswere cloned (FIG. 2B), one of which (ΔTPR) contained the central and UCSdomains, while the other (UCS) contained the UCS domain only.Small-scale baculovirus infections were performed using these threeTgUNC constructs, and expression and solubility of TgMyoA weredetermined by Western blotting. Total (T) and soluble (S) fractions wereprobed with either anti-FLAG (top panel) for TgMyoA heavy chain oranti-Myc (bottom panel) for TgUNC and its truncations (FIG. 8). Theresults showed that the TPR domain was dispensable to obtain solublemyosin, while the UCS domain alone produced very little soluble myosin.

To determine the functionality of the TgMyoA, large-scale infections andpurifications were done with each of the TgUNC constructs, co-expressedwith TgMyoA heavy chain and both light chains. All three infectionsyielded protein, but the yields were reduced for the two shorter TgUNCconstructs. Per 10⁹ Sf9 cells, full-length TgUNC yielded ˜1.5 mg TgMyoA,ΔTPR ˜0.35 mg TgMyoA, and UCS only ˜0.1 mg TgMyoA. In the in vitromotility assay, the motor expressed with either full-length TgUNC orΔTPR generated similar actin sliding speeds (3.4±0.7 μm/s and 3.1±0.5μm/s, respectively; see FIG. 9). In contrast, speeds decreased more thanfour-fold, to 0.7±0.2 μm/s, when TgMyoA was expressed in the presence ofthe UCS domain alone (FIG. 9).

DISCUSSION

It has now been shown that the class XIVa myosin motor TgMyoAsimultaneously binds two light chains: the well-established TgMLC1 and asecond light chain called TgELC1 (6,8). TgMyoA thus has a domainstructure more similar to conventional myosins than previouslyappreciated. The results of the studies outlined above herein indicatethat TgMyoA has a fairly conventional lever arm with two bound lightchains, formed by TgELC1 and TgMLC1 binding to adjacent sites at the Cterminus of the TgMyoA heavy chain. The lever arm functions to amplifysmall changes at the active site into the larger motions necessary topropel actin at fast speeds, and the length of the lever arm dictatesthe speed. TgMyoA with only TgMLC1 bound moved actin at half the speedof TgMyoA with both TgMLC1 and TgELC1 bound (1.5 μm/s vs. 3.4 μm/s).Consistent with the functional data, reciprocal co-immunoprecipitationsshowed that the TgMyoA heavy chain simultaneously bound both types oflight chains, establishing that TgMLC1 and TgELC1 bind tonon-overlapping sites on the heavy chain.

Role of TgMyoA Light Chains—

The results of the studies indicate that TgELC1 is a bona fide subunitof TgMyoA. The in vitro motility data show that TgMyoA moves actin atthe same speed in the presence or absence of calcium, and does notsupport the idea that calcium binding directly regulates this motor.Increased levels of intracellular calcium within the parasite mayinstead trigger signaling pathways that lead to increased association ofTgELC1 with TgMyoA.

Motor Activity—

The steady-state ATPase assay showed that TgMyoA has a low affinity(i.e., a high Km) for actin in the presence of MgATP, as expected for asingle-headed motor. The extrapolated maximal ATPase rate of ˜80 sec-1gives a total time per cycle of MgATP hydrolysis of ˜12 msec. From themeasured unitary step-size (duni) of 5.3 nm (8) and a speed of actinmovement of ˜3.4 μm/s (v), the time the motor spends strongly attachedto actin is ˜1.5 msec (ton=duni/v). This is a small percentage of thetotal cycle time, and thus this motor has a low duty cycle, as do mostmotors designed for speed.

Proper Folding of TgMyoA Heavy Chain Requires a Parasite-Specific MyosinCo-Chaperone—

A main finding of the studies described herein was that a T. gondiimyosin co-chaperone is required to properly fold TgMyoA heavy chain inthe baculovirus/Sf9 insect cell expression system. This was the firstdemonstration that functional class XIV myosin from an apicomplexanparasite can be expressed in a heterologous system. While TgMyoA can bepurified directly from the parasite, the yields are low, making itdifficult to rigorously characterize the parasite-derived motor complex.With the heterologous expression system described herein, yields of 1 mgwere readily attainable from 1×10⁹ infected Sf9 cells (200 ml culture),comparable to yields obtained with myosins that do not requirecoexpression with an exogenous chaperone. Importantly, the speed atwhich expressed TgMyoA with two bound light chains moves actin in an invitro motility assay (˜3.4 μm/s) is close to the speed of the motorcomplex isolated from parasites (˜5 μm/s) (8,12). The difference in thetwo values may be simply due to differences in the way the speeds werecalculated. In studies described herein, a tracking program was usedthat calculates speeds of hundreds of moving filaments withoutuser-bias, while prior studies tracked individual filaments manually.

The TPR Domain of TgUNC is not Required for its Chaperone Activity—

TgUNC has all three domains found in the canonical Unc45 protein, i.e.,an N-terminal TPR domain, a central domain, and a C-terminal UCS domainthat binds to myosin (see FIG. 2B). It has now been shown that theN-terminal TPR domain of TgUNC is not necessary to obtain functionalTgMyoA in Sf9 cells. Both the myosin-binding UCS domain and the centraldomain are, however, required.

The long-sought goal of obtaining milligram quantities of TgMyoA has nowbeen reached, which for the first time makes high-throughput screeningfor drugs against this unusual, virulence-associated motor possible.Furthermore, the results of these studies supports a conclusion thatgenomes of other apicomplexan parasites encode homologs of TgUNC, andthat the approach described herein may be used for the expression offunctional class XIV myosins from other apicomplexan parasites ofmedical and/or veterinary importance.

REFERENCES

-   1. Foth, B. J., et al, (2006) Proc. Natl. Acad. Sci. USA 103,    3681-3686.-   2. Meissner, M., et al., (2002) Science 298, 837-840.-   3. Sheffield, H. G., and Melton, M. L. (1968) J. Parasitol. 54,    209-226.-   4. Gaskins, E., et al., (2004) J. Cell Biol. 165, 383-393.-   5. Frenal, K., et al., (2010) Cell Host Microbe. 8, 343-357.-   6. Nebl, T., et al., (2011) PLoS Pathog. 7, e1002222.-   7. Heintzelman, M. B., and Schwartzman, J. D. (1997) J. Mol. Biol.    271, 139-146.-   8. Herm-Gotz, A., et al., (2002) EMBO J. 21, 2149-2158.-   9. Bement, W. M., and Mooseker, M. S. (1995) Cell Motil.    Cytoskeleton 31, 87-92.-   10. Kinose, F., et al., (1996) J. Cell Biol. 134, 895-909.-   11. Hettmann, C., et al., (2000) Mol. Biol. Cell 11, 1385-1400.-   12. Heaslip, A. T., et al., (2010) PLoS Pathog. 6, e1000720.-   13. Leung, J. M., et al., (2014) PLoS One 9, e85763.-   14. Hakansson, S., et al., (1999) Mol. Bio. Cell 10, 3539-3547.-   15. Moore, J. R., et al., (2004) J. Muscle Res. Cell Motil 0.25,    29-35.-   16. Warshaw, D. M., et al., (2000) J. Biol. Chem. 275, 37167-37172.-   17. Tyska, M. J., and Warshaw, D. M. (2002) Cell Motil. Cytoskeleton    51, 1-15.-   18. Trybus, K. M. (1994) J. Biol. Chem. 269, 20819-20822.-   19. Pato, M. D., et al., (1996) J. Biol. Chem. 271, 2689-2695.-   20. De La Cruz, E. M., et al., (1999) Proc. Natl. Acad. Sci. USA 96,    13726-13731.-   21. Trybus, K. M., et al., (1999) J. Biol. Chem 0.274, 27448-27456.-   22. Wang, F., et al., (2000) J. Biol. Chem. 275, 4329-4335.-   23. Wells, A. L., et al., (1999) Nature 401, 505-508.-   24. Yang, Y., et al., (2005) J. Biol. Chem. 280, 32061-32068.-   25. Chow, D., et al., (2002) J. Biol. Chem. 277, 36799-36807.-   26. Srikakulam, R., and Winkelmann, D. A. (1999) J. Biol. Chem. 274,    27265-27273.-   27. Srikakulam, R., and Winkelmann, D. A. (2004) J. Cell Sci. 117,    641-652.-   28. Barral, J. M., et al., (2002) Science 295, 669-671.-   29. Liu, L., et al., (2008) J. Biol. Chem. 283, 13185-13193.-   30. Srikakulam, R., et al., (2008) PLoS One 3, e2137.-   31. Scheufler, C., et al., (2000) Cell 101, 199-210.-   32. Hutagalung, A. H., et al., (2002) J. Cell Sci. 115, 3983-3990.-   33. Trybus, K. M. (2000) Methods 22, 327-335.-   34. Philo, J. S. (2000) Anal. Biochem. 279, 151-163.-   35. Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333.-   36. Beckingham, K. (1991) J. Biol. Chem. 266, 6027-6030.-   37. Bergman, L. W., et al., (2003) J. Cell Sci. 116, 39-49.-   38. Bosch, J., et al., (2007) J. Mol. Biol. 372, 77-88.-   39. Turley, S., et al., (2013) Mol. Biochem. Parasitol. 190, 56-59.-   40. Treeck, M., et al., (2011) Cell Host Microbe 10, 410-419.-   41. Sakamoto, T., et al., (2003) J. Biol. Chem. 278, 29201-29207.-   42. Douse, C. H., et al., (2012) J. Biol. Chem. 287, 36968-36977.-   43. Lee, C. F., et al., (2011) Structure 19, 397-408.-   44. Shi, H., and Blobel, G. (2010) Proc. Natl. Acad. Sci. USA 107,    21382-21387.-   45. Mishra, M., et al., (2005) Eukaryot. Cell 4, 567-576.-   46. Ni, W., et al., (2011) J. Cell. Sci. 124, 3164-3173.-   47. Andenmatten, N., et al., (2013) Nat. Methods 10, 125-127.-   48. Egarter, S., et al., (2014) PLoS One 9, e91819.-   49. Corpet F (1988) Nucleic Acids Research November 25; 16(22):    10881-10890.-   50. Pearson W R, et al., (1997) Genomics 46(1):24-36.-   51. Lee C F, et al. (2011) Structure 19(3):397-408.-   52. Andrade M A, et al., (2001) J Mol Biol 309(1):1-18.

Although several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto; the invention may be practiced otherwise than asspecifically described and claimed. The present invention is directed toeach individual feature, system, article, material, and/or methoddescribed herein. In addition, any combination of two or more suchfeatures, systems, articles, materials, and/or methods, if suchfeatures, systems, articles, materials, and/or methods are not mutuallyinconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified, unless clearly indicated to the contrary.

All references, patents, patent applications, and non-patentpublications that are cited or referred to in this application areincorporated in their entirety herein by reference.

What is claimed is:
 1. A method of producing a functional class XIVmyosin polypeptide, the method comprising, co-expressing three or morepolynucleotides in an expression-system cell, wherein the three or morepolynucleotides comprise a class XIV heavy chain polypeptide-encodingpolynucleotide, a first myosin light chain polypeptide-encodingpolynucleotide, and a parasite co-chaperone polypeptide-encodingpolynucleotide, wherein the three or more polynucleotides areco-expressed in the cell under conditions suitable to produce afunctional class XIV myosin polypeptide comprising the class XIV heavychain polypeptide and the first myosin light chain polypeptide.
 2. Themethod of claim 1, wherein the class XIV heavy chainpolypeptide-encoding polynucleotide, the parasite co-chaperonepolypeptide-encoding polynucleotide, and the first myosin light chainpolypeptide-encoding polynucleotide are each independently selected froma Toxoplasma, Plasmodium, Neospora, Sarcocystis, Eimeria, orCryptosporidium class XIV heavy chain polypeptide-encodingpolynucleotide or a functional variant thereof; a parasite co-chaperonepolypeptide-encoding polynucleotide or a functional variant thereof; anda myosin light chain polypeptide-encoding polynucleotide or a functionalvariant thereof, respectively.
 3. (canceled)
 4. The method of claim 1,wherein co-expressing comprises co-infecting the expression-system cellwith one or more expression vectors each comprising one or more of thethree or more polynucleotides.
 5. The method of claim 4, wherein the oneor more expression vectors is a viral expression vector.
 6. The methodof claim 4, wherein co-infecting the expression-system cell comprisesco-infecting with one or more expression vectors comprising one or moreof the class XIV heavy chain polypeptide-encoding polynucleotideoperably linked to an independently selected promoter, the first myosinlight chain polypeptide-encoding polynucleotide operably linked to anindependently selected promoter, and the parasite co-chaperonepolypeptide-encoding polynucleotide operably linked to an independentlyselected promoter.
 7. The method of claim 4, wherein co-infecting theexpression-system cell comprises co-infecting with a first expressionvector comprising the class XIV heavy chain polypeptide-encodingpolynucleotide operably linked to an independently selected promoter, asecond expression vector comprising the first myosin light chainpolypeptide-encoding polynucleotide operably linked to an independentlyselected promoter, and a third expression vector comprising the parasiteco-chaperone polypeptide-encoding polynucleotide operably linked to anindependently selected promoter.
 8. The method of claim 4, wherein theone or more of the expression vectors additionally comprises one or morepolynucleotides that encode: a myosin light chain-1 (MLC1) polypeptideor functional variant thereof, a tail domain interacting protein (MTIP)or functional variant thereof, an essential light chain-1 (ELC1)polypeptide or functional variant thereof, calmodulin or a functionalvariant thereof, or a glideosome associated protein-45 (GAP45) or afunctional variant thereof.
 9. The method of claim 4, wherein one ormore of the expression vectors additionally comprise at least onepolynucleotide sequence that encodes a detectable label. 10-11.(canceled)
 12. The method of claim 1, wherein the parasite co-chaperonepolypeptide-encoding polynucleotide sequence comprises aUNC-45/Cro1/She4p (UCS) chaperone polynucleotide sequence, or functionalvariant thereof.
 13. The method of claim 1, wherein the parasiteco-chaperone polypeptide is derived from the sequence of a toxoplasmagondii UCS-45 homolog, a Toxoplasma UNC (TgUNC) polypeptide, or afunctional variant thereof. 14-18. (canceled)
 19. The method of claim 1,wherein the parasite co-chaperone is derived from the sequence of aPlasmodium falciparum UCS-45 homolog, a Plasmodium falciparum UNC(PfUNC) polypeptide, or a functional variant thereof. 20-21. (canceled)22. The method of claim 19, wherein the PfUNC functional variant is atruncated PfUNC polypeptide.
 23. (canceled)
 24. The method of claim 1,wherein the expression system is a baculovirus/insect cell expressionsystem. 25-31. (canceled)
 32. The method of claim 1, wherein the firstmyosin light chain polypeptide is a regulatory light chain (MLC1)polypeptide sequence derived from a Toxoplasma gondii regulatory lightchain polypeptide sequence, or derived from a Plasmodium falciparumregulatory light chain polypeptide sequence. 33-36. (canceled)
 37. Themethod of claim 1, further comprising isolating the expressed functionalclass XIV myosin polypeptide.
 38. The method of claim 1, furthercomprising assaying the function of the expressed functional class XIVmyosin polypeptide.
 39. (canceled)
 40. The method of claim 1, furthercomprising co infecting the expression-system cell with an expressionvector comprising a second myosin light chain encoding polynucleotide;and co-expressing the class XIV heavy chain polynucleotide, the firstand second myosin light chain polynucleotides, and the parasiteco-chaperone polynucleotide under conditions suitable to produce afunctional class XIV myosin polypeptide comprising the class XIV heavychain polypeptide and the first and second myosin light chainpolypeptides. 41-46. (canceled)
 47. A functional class XIV myosinpolypeptide prepared by the method of claim
 1. 48. (canceled)
 49. Amethod of determining an activity of a class XIV myosin polypeptide, themethod comprising, (a) preparing a functional class XIV myosinpolypeptide with a method comprising co-expressing three or morepolynucleotides in an expression-system cell, wherein the three or morepolynucleotides comprise a class XIV heavy chain polypeptide-encodingpolynucleotide, a first myosin light chain polypeptide-encodingpolynucleotide, and a parasite co-chaperone polypeptide-encodingpolynucleotide, wherein the three or more polynucleotides areco-expressed in the cell under conditions suitable to produce afunctional class XIV myosin polypeptide comprising the class XIV heavychain polypeptide and the first myosin light chain polypeptide; (b)assaying an activity of the prepared functional class XIV myosinpolypeptide; and (c) assessing the results of the assay as adetermination of activity in the functional class XIV myosinpolypeptide. 50-51. (canceled)
 52. A method of identifying a candidatecompound to inhibit a parasite that expresses a class XIV myosinpolypeptide, the method comprising, (a) preparing a functional class XIVmyosin polypeptide with a method comprising, co-expressing three or morepolynucleotides in an expression-system cell, wherein the three or morepolynucleotides comprise a class XIV heavy chain polypeptide-encodingpolynucleotide, a first myosin light chain polypeptide-encodingpolynucleotide, and a parasite co-chaperone polypeptide-encodingpolynucleotide, wherein the three or more polynucleotides areco-expressed in the cell under conditions suitable to produce afunctional class XIV myosin polypeptide comprising the class XIV heavychain polypeptide and the first myosin light chain polypeptide; (b)contacting the functional prepared class XIV myosin polypeptide with acandidate compound under conditions suitable to determine an activity ofthe class XIV myosin polypeptide; (c) determining the activity of thefunctional class XIV myosin polypeptide; and (d) comparing thedetermined activity with a control activity determination, wherein adecrease in the determined activity in the contacted functional classXIV myosin polypeptide compared to the control activity determinationidentifies the compound as a candidate compound to inhibit a parasitethat expresses the functional class XIV myosin polypeptide. 53-61.(canceled)